“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae

Abstract

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

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MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, June 2004, p. 187–206 Vol. 68, No. 2
1092-2172/04/$08.00⫹0 DOI: 10.1128/MMBR.68.2.187–206.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae†
Joseph V. Gray,
1
* Gregory A. Petsko,
2
Gerald C. Johnston,
3
Dagmar Ringe,
2
Richard A. Singer,
4
and Margaret Werner-Washburne
5
Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU,
United Kingdom
1
; Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham,
Massachusetts 02454-9110
2
; Department of Microbiology and Immunology
3
and Department of
Biochemistry and Molecular Biology,
4
Dalhousie University, Halifax, Nova Scotia B3H 1X5,
Canada; and Biology Department, University of New Mexico,
Albuquerque, New Mexico 87131
5
INTRODUCTION .......................................................................................................................................................188
QUIESCENCE IN YEAST.........................................................................................................................................188
Operational Definition of Quiescence..................................................................................................................188
Characteristics of Quiescent Cells .......................................................................................................................189
Cell Quiescence Cycle.............................................................................................................................................190
Mutants Defective in the Cell Quiescence Cycle................................................................................................190
ENTRY INTO QUIESCENCE ..................................................................................................................................191
TOR Pathway ..........................................................................................................................................................191
Protein Kinase C Pathway.....................................................................................................................................192
Protein Kinase A Pathway.....................................................................................................................................192
Snf1p Pathway.........................................................................................................................................................193
An Emerging Signaling Network ..........................................................................................................................193
Working Model for the Regulation of Entry into Quiescence..........................................................................194
MAINTENANCE OF VIABILITY IN QUIESCENCE ...........................................................................................194
Genes Required for Maintaining Viability..........................................................................................................194
“Essential” Genes That Are Not Required for Viability ...................................................................................195
Control of Gene Expression ..................................................................................................................................195
Translation...............................................................................................................................................................196
Protein Turnover and Covalent Modification of N Termini.............................................................................196
Autophagy ................................................................................................................................................................197
Metabolism ..............................................................................................................................................................198
Redox Homeostasis.................................................................................................................................................198
Aging versus Maintenance of Viability ................................................................................................................198
EXITING FROM QUIESCENCE.............................................................................................................................199
Sensing Nutrients ...................................................................................................................................................199
Transcriptional Changes........................................................................................................................................199
Proteome Changes ..................................................................................................................................................200
Exit Mutants............................................................................................................................................................200
Gcs1 and Vesicular Traffic ....................................................................................................................................200
CONCLUSIONS AND PERSPECTIVES.................................................................................................................201
ACKNOWLEDGMENTS ...........................................................................................................................................202
REFERENCES ............................................................................................................................................................202
“Beloved, may your sleep be sound
That have found it where you fed”
William Butler Yeats (Lullaby)
INTRODUCTION
All living cells appear to be capable of exiting the normal cell
cycle (proliferating state) and entering an alternative (resting)
state termed quiescence or G
(0)
. Quiescent microbes are
thought to represent about 60% of the biomass on Earth and
are doubtless the seeds of microbial life in nature. Further-
more, most eukaryotic cells, whether they exist as single-celled
or multicellular organisms, spend the majority of their natural
lives in a quiescent state (87). Quiescent cells of both prokary-
otic and eukaryotic microorganisms can survive for long peri-
ods—sometimes years—without added nutrients, a feat of as-
tonishing resilience (167).
Beyond contributing to a more rounded view of the life cycle
of cells, understanding quiescence has other potentially signif-
icant implications. A deeper understanding of the conserved
mechanisms underlying entry into, survival in, and exit from
quiescence in eukaryotes may aid the development of novel or
supplementary immunosuppressants and anticancer therapies
and is also likely to provide significant insights into such di-
* Corresponding author. Mailing address: Division of Molecular
Genetics, Faculty of Biomedical and Life Sciences, University of Glas-
gow, Anderson College, 56 Dumbarton Rd., Glasgow G11 6NU,
United Kingdom. Phone: (0)141-330-5114/6235. Fax: (0)141-330-4878.
E-mail: J.Gray@bio.gla.ac.uk.
† This paper is dedicated to the memory of Ira Herskowitz and
Helmut Ruis.
187

verse processes as aging (50) and neurodegenerative diseases
(139). The discovery of variations on a common theme may
allow the development of novel antipathogenic agents. Fur-
ther, most of the world’s microorganisms have yet to be cul-
tured. Among these organisms are likely to be many novel
microbes, predominantly in a quiescent state, that can produce
medically useful natural products or whose study will provide
new insight into evolution, development, and ecology. An un-
derstanding of how to stimulate these microbes to exit from
quiescence may aid the culturing of such organisms.
Although we ultimately seek to understand aspects of qui-
escence shared among all eukaryotes, we focus this review on
quiescence in the budding yeast Saccharomyces cerevisiae.We
restrict our focus for several reasons. First, S. cerevisiae is one
of the best-studied eukaryotes and is tractable to all levels of
experimental analysis. Second, because of the conservation of
basic cellular processes among eukaryotes, the study of quies-
cence in yeast is likely to illuminate the equivalent mechanisms
and states in many if not all other eukaryotes and possibly
prokaryotes as well. Indeed, even mammals possess orthologs
of the apparent yeast regulators (see below), such as the TORs,
protein kinases A and C, and Snf1p. This is not surprising. The
ability of microbes and our microbial ancestors to enter qui-
escence and thereby maintain viability when starved is likely to
have been essential to their survival. A strong selective pres-
sure has doubtless acted to maintain the ability to enter into,
survive in, and exit from quiescence over evolutionary time.
Third, quiescent yeast and quiescent mammalian cells share a
number of salient characteristics such as unreplicated genomes
(121); characteristically condensed chromosomes (113), re-
ferred to as G
(0)
chromosomes; increased rates of autophagy;
and reduced rates of translation (see below for details). Fur-
thermore, both yeast and mammalian cells respond similarly to
rapamycin, an immunosuppressant drug for humans, which
inhibits the proliferation of both yeast and mammalian cells
and drives each into a state similar to their respective quiescent
state (135). Even though entry of cells into and exit of cells
from quiescence in metazoan bodies is normally regulated by
positional and developmental cues, mammalian cells share
with yeast cells the ability to respond to starvation by entering
quiescence-like states (91). Finally, with the exploitation of the
genome sequence, the new technologies available to study
yeast further ensures that this small eukaryote will be central to
unlocking the secrets of the quiescent state.
Our knowledge of quiescence in any organism including
yeast is fragmented, and the mechanisms that regulate entry
into, maintenance of, and exit from quiescence are, at best,
poorly understood. Historically, one major factor limiting the
study of quiescent cells has been their very modest life-style:
classical cell biological, physiological, and biochemical assays
detected little or no activity in these cells. Furthermore, the
application of genetics to the study of quiescence has been
limited (see below for further considerations of this point). As
a result, many researchers have long suspected that quiescent
cells are difficult to study (historically correct), do not repre-
sent a distinct phase of cell (probably inaccurate), do not do
anything (inaccurate), or are either uninteresting or dead (very
inaccurate). Even now, understanding the mechanisms by
which a cell transits between the proliferating and quiescent
states and the way in which these states differ presents a for-
midable challenge. As we shall argue, switching between active
proliferation and quiescence is likely to involve the wholesale
reprogramming of regulatory networks and the remodeling of
most if not all intracellular structures and processes.
The “holy grail” for researchers working on quiescence is to
define a core quiescence program that prevents cell growth and
proliferation, that confers on cells the ability to survive better
under adverse conditions, and that allows a rapid transition
back to the proliferating state when conditions again become
favorable. Here, as a starting point, we present an overview of
the current understanding of quiescence in yeast.
Almost 400 papers dealing with some aspect of quiescence in
yeast have been published since our last major review of this
topic (167, 168). We cannot completely cover this literature,
but we provide a list of all the papers that we identified in this
area on our website (http://biology.unm.edu/biology/maggieww
/SPreview.htm). Some literature not covered herein has been
discussed in another recent and shorter review of stationary-
phase yeast cultures, i.e., those containing quiescent cells (59).
QUIESCENCE IN YEAST
Operational Definition of Quiescence
Quiescent yeast cells are commonly obtained in the labora-
tory by growing liquid cultures to saturation in rich media,
usually for 5 to 7 days at 30°C (Fig. 1). The term “stationary
phase” has been used to describe the state of saturated liquid
cultures and the state of the constituent cells. We propose a
revision of this nomenclature, such that “stationary phase” is
used to refer to the state of a saturated culture and the term
“quiescence” is used to refer to the state of the constituent
cells in such a saturated culture (Fig. 1). It is not known if all
cells in a stationary-phase culture are quiescent, but we assume
that a substantial proportion are, including the daughter cells
that were produced during the final doublings in the post-
diauxic phase of culture growth.
We currently define the reference quiescent state in yeast as
the state of the cell brought about by growth of a liquid culture
of cells to saturation in rich media (yeast-peptone-dextrose
[YPD]). Once this and other quiescence-like states of yeast
have been more closely examined and compared, this defini-
tion of quiescence will doubtless become more refined.
The path by which such a culture of cells reaches saturation
is not simple (Fig. 1). Initially, the constituent cells derive their
energy from fermentation, the process by which glucose is
preferentially metabolized via glycolysis to form nonferment-
able carbon compounds, particularly ethanol. During the ex-
ponential or logarithmic growth phase, the culture grows rap-
idly (and the constituent cells proliferate with an average
doubling time of approximately 90 min at 30°C) until glucose is
exhausted in the medium. At this point, termed the diauxic
shift, the culture ceases rapid growth while the constituent cells
readjust their metabolism to utilize the nonfermentable carbon
sources still present in the medium. After the diauxic shift, the
cells in the culture undergo one or two very slow doublings
over a period of days before finally ceasing proliferation after
the depletion of ethanol and other nonfermentable carbon
sources (88). At this point, the culture is in stationary phase
and most, if not all, of the constituent cells are quiescent (167).
188 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

All cultures having passed the diauxic shift are often (mis-
takenly, we believe) classified as being in stationary phase and
the constituent cells thus in quiescence. We favor drawing a
clear distinction between cells found in the post-diauxic shift
state of a culture prior to saturation and quiescent cells found
in a saturated culture. This distinction may be more than se-
mantic: post-diauxic shift cells have acquired many, but not all,
of the characteristics of quiescent cells and continue to prolif-
erate, albeit very slowly. Entry of cells into quiescence is best
viewed as a stepwise process in liquid culture, with various
characteristics of the quiescent state acquired either at the
diauxic shift or on final cessation of proliferation (see below).
Although our operational definition of quiescence (the state
of cells in liquid cultures grown to saturation in rich media)
focuses attention on a specific reference state, the definition
may be overly restrictive. It is clear that many of the cells in a
colony growing on the surface of solid rich media are also in
quiescence (108). However, because of the immobility of yeast
cells on solid surfaces, distinct microenvironments develop
within a colony, allowing multiple subpopulations of cells to
coexist. This is in stark contrast to a culture of cells in agitated
liquid medium, where every cell experiences the same, homo-
geneous environment. The response of a colony of cells to
starvation is thus likely to be significantly more complicated
than, although closely related to, that of cells in liquid cultures.
Our operational definition of quiescence may also be overly
restrictive because yeast cells enter somewhat stable nonpro-
liferating states when rapidly starved for nitrogen, sulfur, or
phosphate or when transferred directly to water (149). Indeed,
spores, formed when diploid cells are starved for nitrogen in
the presence of a poor carbon source, are also in a quiescence-
like state: they can remain viable for many years—possibly
centuries (99). Because the relationships between these
“other” nonproliferating cell states and quiescence are not yet
known, we do not discuss these states further here.
It should be noted that not all starvations cause entry into
viable nonproliferating states. For example, starvation of ino-
sitol auxotrophs for inositol causes rapid cell death (“inositol-
less death”), apparently by disrupting the ordered growth of
the cell (78). In addition, most laboratory strains lose viability
relatively quickly when grown to saturation in synthetic, de-
fined medium (7). It thus appears that entry into quiescence
(and quiescence-like states) is a programmed response to spe-
cific environmental changes and does not occur simply by de-
fault.
Characteristics of Quiescent Cells
The relationship between the state we term quiescence or
G
(0)
in yeast and the so-called G
(0)
state of nonproliferating,
terminally differentiated mammalian cells such as neurons and
fibroblasts remains to be established, but it is likely that qui-
escent yeast will prove to be an important model for under-
standing the G
(0)
state of multicellular eukaryotes, as it has
been for so many aspects of the proliferative cell division cycle.
Quiescent yeast cells display numerous specific characteris-
tics that differentiate them from proliferating cells: they do not
proliferate; they fail to accumulate mass and volume; they are
arrested as unbudded cells (121); the overall transcription rate
is three to five times lower than in logarithmic-phase cultures
(20); they have a requirement for translation from internal
initiation sites (internal ribosome entry site) (108); expression
of a subset of genes is severely repressed, e.g., those encoding
ribosomal proteins; expression of a subset of genes is strongly
induced, e.g., SNZ1, HSP26, and UBI4 (168); mRNA degra-
dation is inhibited (73); overall protein synthesis is reduced to
approximately 0.3% of the rate found in logarithmically grow-
ing cultures (47); chromosomes are condensed [G
(0)
chromo-
somes, (113)]; autophagy (the process of engulfment of the
cytoplasm into lipid vesicles for delivery to the vacuole for
degradation) is induced (103); cells develop thickened cell
walls and are more resistant than are proliferating cells to
digestion by zymolyase and to treatment with certain toxic
drugs (30); and cells are more thermotolerant and osmotoler-
ant than are their proliferating counterparts (114).
Perhaps surprisingly, quiescent yeast cells are capable of
responding to environmental signals in addition to the pres-
ence of carbon. Irradiation, heat shock, and treatment with
chemicals such as methylmethane sulfonic acid and certain
toxins can induce the expression of similar genes in both qui-
escent and proliferating yeast (68) (M. Werner-Washburne,
unpublished data), as can oxidative stress (26).
The above characteristics begin to define a set of landmarks
that can be used to identify and characterize cells in quiescence
and suggest that the quiescent and proliferating states are
distinct. Furthermore, a variety of mutants, including ubi4,
ard1, and some alleles of bcy1, are known to selectively or
specifically die when starved, supporting the notion that the
proliferating and quiescent states are distinct. However, per-
haps the most compelling argument that quiescence should be
FIG. 1. Relationship between the state of a culture of yeast cells
growing to saturation in rich medium (YPD) and the state of the
constituent cells. When yeast cells are inoculated into rich medium
containing glucose, the cells proliferate rapidly using fermentation and
the density of the culture (reflected in optical density at 600 nm
[OD
600
]) increases logarithmically with time (log phase). When glu-
cose is consumed in the culture at the diauxic shift (after approximately
1 day), the cells cease rapid cell proliferation and readjust their me-
tabolism from fermentation to respiration to utilize other carbon
sources present in the medium. In the resulting post-diauxic shift state
of the culture, constituent cells proliferate very slowly. When external
carbon sources are exhausted, the culture reaches saturation (at ap-
proximately 5 to 7 days postinoculation) and the constituent cells cease
proliferation and enter the quiescent state.
VOL. 68, 2004 QUIESCENCE IN YEAST 189

considered a distinct developmental state comes from the
gcs1⌬ mutant, which proliferates normally in the presence of
food, enters quiescence when grown to stationary phase, and
maintains viability in quiescence normally but is conditionally
defective in exiting from quiescence and returning to active
proliferation when nutrients are restored. gcs1⌬ mutants are
cold sensitive only for exiting from quiescence (34, 35).
Cell Quiescence Cycle
The process of entering into quiescence has traditionally
been represented as a reversible reaction, with exit from qui-
escence being simply the reverse of entry. However, we think
that this view is too simplistic. Entry into quiescence is trig-
gered when a proliferating cell senses carbon limitation. In
contrast, exit is triggered by a different state of the cell (qui-
escent) sensing the presence of a carbon source. There is no
reason to believe that the processes of entry into and exit from
quiescence share any common intermediate states of the cell
(see “Exiting from quiescence” below).
We therefore propose a revision of this traditional view in
which entry into, survival in, and exit from the quiescent state
can be regarded as a developmental process that, by analogy to
the proliferative cell cycle, can be called the cell quiescence
cycle (see Fig. 2). In this view, entry into and exit from quies-
cence are distinct processes. As with the proliferative cell di-
vision cycle, passage around one complete round of the quies-
cence cycle returns the starting cell, to a first approximation,
back to its starting state. In reality, each turn of either cycle
changes the state of the starting cell: in the case of the cell
cycle, the mother cell becomes one generation older (i.e., has
reduced replicative capacity); in the case of the quiescence
cycle, the cell also becomes older, again with respect to loss of
replicative capacity (2). The cell cycle results in a doubling of
cell the number, whereas the quiescence cycle does not.
The cell division cycle and the cell quiescence cycle intersect
at the G
1
phase (Fig. 2). In this phase, a cell can enter either
the cell division cycle or the quiescence cycle (58, 167). In the
presence of ample food supplies (and other conditions permit-
ting), a G
1
cell passes START (58) and enters the proliferative
cell cycle. The subsequent removal of nutrients does not gen-
erally hamper completion of the ongoing cell cycle, which is
driven by internally controlled fluctuations in cyclin-dependent
protein kinase activity (104). In the absence of a sufficient
carbon source, a G
1
cell fails to pass START and enters the
quiescence cycle. Unlike the proliferative cell cycle, the quies-
cence cycle does not turn under its own steam but cycles with
changes in the environment: the lack of nutrients triggers entry
into quiescence; the resupply of nutrients triggers exit.
Mutants Defective in the Cell Quiescence Cycle
The isolation of mutants defective in key transitions of the
cell quiescence cycle is worthy of some consideration. To date,
the most frequently reported class of relevant mutants appears
to lose viability when cultured to stationary phase: the mutant
cells lose the ability to form colonies when subsequently trans-
ferred back to nutrient-rich media. In many cases, this inter-
pretation may be naı´ve. Three subclasses of “stationary-phase”
mutants are likely to exist, all of which may be defective in one
or more transition of the cell quiescence cycle.
The first subclass includes entry mutants, i.e., those that fail
to enter quiescence properly. Such mutants would be expected
to die when starved, given the likelihood that successful entry
into quiescence is required for cells to remain viable when
starved. The second subclass includes maintenance mutants,
i.e., those that successfully enter quiescence (acquire all the
key characteristics of quiescent cells) but are unable to main-
tain viability in that state. Two subclasses of these maintenance
mutants are likely to exist: those inherently required for via-
bility in the quiescent state itself, and those specifically defec-
tive in surviving when starved. The third subclass includes exit
mutants, i.e., those that enter quiescence and remain viable
normally but are specifically unable to return to the prolifer-
ating state when nutrients again become available. The cells of
such mutants are viable when starved but are unable to gen-
erate CFU on replating.
In only a handful of cases have the above distinctions been
entertained. Thus, mutants reported to be lose viability in
FIG. 2. The cell quiescence cycle and its relationship to the cell
division cycle. The cell quiescence cycle is the process by which nutri-
ent limitation (e.g., carbon starvation in our reference case) causes exit
from active proliferation (the cell division cycle) and triggers entry into
the stable nonproliferating state, quiescence/G
(0)
. Only after a favor-
able change in nutrient availability will a turn of the quiescence cycle
be completed, since nutrient availability triggers exit from quiescence/
G
(0)
. The cell quiescence cycle and the cell division cycle intersect at
the G
1
phase, where a cell has not yet committed to the cell division
cycle. In the presence of sufficient nutrients and with no other influ-
ences, a G
1
cell will pass START, after which it is committed to
completing a turn of the cell division cycle with production of a daugh-
ter cell. Slow depletion of an essential nutrient such as carbon will
allow the completion of an ongoing cell division cycle but will not allow
passage through START. In this case of insufficient nutrient availabil-
ity, the cell will enter the cell quiescence cycle.
190 GRAY ET AL. M
ICROBIOL.MOL.BIOL.REV.

stationary-phase cultures may in reality fall into any one of the
above classes when properly analyzed. Much of the published
literature on “stationary-phase”/quiescent mutants should thus
be approached with some caution. A second limitation of the
published literature is that many investigators have assumed,
incorrectly, that any post-diauxic shift culture is in stationary
phase, with its constituent cells in quiescence (see above).
Subclassification of “stationary-phase” mutants is possible.
First, many characteristics of quiescent cells are known, and,
together, these constitute a reference set of parameters that
can be used to determine (or at least estimate) if mutant cells
cultured to stationary phase enter quiescence successfully. Sec-
ond, cell viability can be assayed independently of the ability to
subsequently proliferate when refed. It has recently been
shown that viability dyes such as methylene blue can be useful
to directly determine the viability of starved cells (80). Fur-
thermore, as outlined above, viable quiescent cells can mount
transcriptional responses to a variety of environmental stresses
and chemical treatments.
It should be borne in mind that a mutant defective in pro-
ducing a protein may show a terminal defect at a stage of the
quiescence cycle distinct from the point at which the protein
acts in wild-type cells. For example, it is conceivable that a
primary defect in fully entering quiescence may not compro-
mise cell viability but, rather, may prevent successful exit after
the stimulation brought about by addition of nutrients. Quies-
cent yeast cells are poised to respond to nutrients, should they
become available, and can do so within seconds of nutrient
resupply. It is likely that a critical property of quiescence is the
ability to exit from that state as quickly as possible once con-
ditions improve.
ENTRY INTO QUIESCENCE
Significant progress toward understanding the mechanisms
regulating entry into quiescence has been made in the last
decade. The relevant gene products have been found in a
variety ways: by studying the response of yeast to the immu-
nosuppressant rapamycin; by identifying temperature-sensitive
mutants that arrest in a quiescence-like state at nonpermissive
temperatures even in the presence of nutrients, e.g., cdc25
(34); and by studying a subset of mutants fortuitously found to
selectively lose viability when cultured to stationary phase.
Here, we discuss the signaling pathways thought to regulate
entry into quiescence: the TOR and protein kinase A (PKA)
pathways, apparent negative regulators of the transition into
quiescence; and the protein kinase C (PKC) and Snf1p path-
ways, apparent positive activators of the transition (Fig. 3).
Our understanding of the signaling networks regulating this
transition is still fragmented, and other key regulators doubt-
less remain to be discovered. We therefore cannot yet tell the
whole story; instead, we summarize a work in progress.
TOR Pathway
The immunosuppressant drug rapamycin inhibits prolifera-
tion of both yeast and mammalian cells. Rapamycin-treated
yeast cells appear to enter a quiescence-like state (3). Treated
haploids arrest as small, unbudded cells with 1’N DNA content
and undergo many of the gene expression changes character-
istic of quiescent cells including repression of the ribosomal
protein genes and induction of UBI4 and HSP26 (57). Rapa-
mycin-treated yeast also synthesize proteins at 50 to 60% of the
level of logarithmically growing cultures, display reduced ac-
tivity of RNA Polymerase I (PolI) and PolIII and high levels of
autophagy (103), and accumulate the storage carbohydrates
glycogen and trehalose.
The cytosolic target of rapamycin is FKBP12, an immu-
nophilin (135). This binary rapamycin-FKBP12 complex binds
to and inhibits the partially redundant proteins Tor1p and
Tor2p when complexed with two other essential proteins,
Lst8p and Kog1p, in the so-called TORC1 complex (89). The
TOR proteins (for “target of rapamycin”) are phosphatidyl-
inositol kinase-related protein kinases (69). Loss of both Tor1p
and Tor2p largely phenocopies rapamycin treatment (3).
Based on these and other observations, Hall and coworkers
have proposed that the TOR proteins function to repress a
quiescence program when nutrients are abundant. They envis-
age that nitrogen or carbon starvation would lead to inactiva-
tion of the TOR pathway, liberation of the quiescence pro-
gram, and consequent entry into quiescence (25, 36, 94, 119,
131, 135).
Some progress has been made in recent years in identifying
the downstream functions of Tor proteins, although direct in
vivo targets of the kinases are still not known. One important
downstream component is Tap42p, which binds to and regu-
lates the catalytic subunits of PP2A protein phosphatases such
as Sit4p, Pph21p, and Pph22p (32, 69). The TORs promote
association of Tap42p with PP2A catalytic subunits when nu-
trients are plentiful. Both rapamycin treatment and transit
through the diauxic shift cause dissociation of Tap42p from its
phosphatase partners (32, 36, 66). Thus, the diauxic shift and
rapamycin act to downregulate TOR function.
Using microarray profiling, a global picture of the gene ex-
pression changes caused by rapamycin treatment (and thus, by
implication, inhibition of the common function of Tor1p and
Tor2p) has emerged (see, e.g., reference 138). Inhibition of
FIG. 3. Summary of the known signaling pathways thought to con-
trol aspects of entry into quiescence. The TOR and PKA pathways are
active in the presence of nutrients and act to repress aspects of quies-
cence. When cells are starved of carbon, both pathways are downregu-
lated. Inactivation of the TORs causes activation (albeit transiently) of
PKC, leading to some characteristics of quiescence such as a remod-
eled cell wall. The Snf1 pathway is inhibited by the presence of fer-
mentable carbon sources such as glucose. When such sources are
depleted, Snf1 is activated and contributes to the switch from fermen-
tative to respiratory metabolism that is essential for entry into quies-
cence.
V
OL. 68, 2004 QUIESCENCE IN YEAST 191

TOR function causes activation of Gln3p and Gat1p transcrip-
tion factors via Tap42p, resulting in induced expression of
nitrogen discrimination pathway (NDP) and carbon discrimi-
nation pathway (CDP) genes, which are normally induced by a
shift from good to poor nitrogen or carbon nutrient sources,
respectively (22). Inhibition of TOR function also causes acti-
vation of Mks1p and consequent induction of Rtg1p- and
Rtg3p-regulated genes, particularly those encoding some of
the Krebs cycle enzymes (147), and activation of Hap2/3/4/5p
with consequent induction of genes encoding other Krebs cycle
enzymes.
Inactivation of the TORs also causes gene expression
changes independently of Tap42p, such as decreased expres-
sion of the ribosomal protein genes and coordinated genes
encoding components of the translational apparatus (120) and
activation of the transcription factors Msn2p and Msn4p, by
promoting their dissociation from 14-3-3 protein anchors in the
cytoplasm. These redundant transcription factors drive the ex-
pression of the stress response element-containing genes,
which are also induced by multiple other environmental stress
such as heat shocks and hyperosmotic shocks (4, 49).
Is inhibition of the TOR pathway important for the forma-
tion of quiescent cells triggered by starvation? Probably. The
phenotype of rapamycin-treated cells and the inactivation of
the pathway on carbon starvation suggests that inhibition of
the TOR pathway is important, if not critical, for entry into
quiescence. In addition, mutants defective in a number of
downstream targets of the TORs, e.g., in autophagy or in the
protein kinase C pathway (see below), die on starvation, sup-
porting a key role for the TORs (77, 80, 154). Unfortunately,
no constitutively activated alleles of TOR1 and TOR2 exist,
precluding a definitive test of this hypothesis.
Inactivation of the TORs may not be sufficient for the for-
mation of truly quiescent cells since rapamycin-treated cells do
not appear identical to quiescent cells. First, rapamycin inhibits
translation by only 50 to 60% (i.e., up to half) of the rate
measured for untreated, logarithmically growing cultures
whereas quiescent cells display 0.3% (i.e., 1/333) of that trans-
lation rate (3, 47, 82). Second, rapamycin-treated cells appear
to continue to accumulate mass and volume, unlike truly qui-
escent cells (3). Finally, rapamycin treatment induces both
NDP and CDP gene expression but carbon limitation induces
only CDP (57).
Whatever the importance of the TORs in defining the qui-
escent state, it is clear that an understanding of their regulation
should inform any model of the elusive nutrient detection
systems that regulate the entry into quiescence. The TORs are
inactivated to some extent at the diauxic shift, by transfer from
good- to poor-quality carbon or nitrogen sources or by starva-
tion for carbon or nitrogen. The TORs are thus responding to
the absence of high-quality nutrient sources as opposed to the
presence of low-quality ones. Many potential regulators of
mTOR (mammalian TOR) have been proposed, including the
possibility that it is a direct sensor of cytoplasmic ATP by virtue
of an unusually high K
m
for ATP (29). How mTOR is regulated
by nutrients and by growth factors is still hotly disputed at the
time of writing, and the identity of the mechanisms that regu-
late the yeast TORs is also unknown.
Protein Kinase C Pathway
The yeast PKC, encoded by PKC1, responds to cell surface
stresses and changes in the actin cytoskeleton during vegetative
proliferation (53, 75, 86). Pkc1p in part regulates a mitogen-
activated protein (MAP) kinase cascade involving the MAP
kinase Mpk1p (23, 65, 83, 84). Mutants lacking BCK1, encod-
ing the MAP kinase kinase kinase that acts in this cascade,
were reported to die rapidly on nitrogen limitation (23, 24).
Based on this observation, it was proposed that the Pkc1p-
MAP kinase pathway may be a nutrient sensor.
It was recently reported that the Pkc1p-MAP kinase path-
way is required for viability on carbon or nitrogen starvation or
growth of a culture to stationary phase (80). However, the
pathway is unlikely to be a nutrient sensor. Rather, it acts
downstream of and is transiently activated by TOR inactivation
(80). Mpk1p is also activated transiently at the diauxic shift
(i.e., concomitant with TOR inactivation) and mpk1⌬ mutants
begin to lose viability at the same point (154). Activation of the
Pkc1p pathway by TOR inhibition occurs by a novel mecha-
nism independent of the Hcs77p and Mid2p sensors required
for detecting cell surface stresses during vegetative prolifera-
tion (53, 67, 79, 124, 160).
The Pkc1p pathway acts, in part, to promote the acquisition
of one key characteristic of quiescent cells on starvation: a
reinforced and remodeled cell surface wall (80). First, mutants
defective in the Pkc1p pathway lyse when starved, and this lysis
is coincident with cell death. Second, starvation or rapamycin
treatment rapidly causes increased resistance to the cell wall-
digesting enzyme zymolyase, and this acquisition of zymolyase
resistance is dependent on Pkc1p. This failure of Pkc1p path-
way mutants to acquire resistance to zymolyase occurs before
cell death, indicating that the pathway is a bona fide positive
regulator of entry into quiescence, which probably acts down-
stream of TOR inactivation.
Curiously, rapamycin treatment alone, even in rich media, is
sufficient to kill mutants defective in the Pkc1p pathway (80).
Thus, mutants lacking components of the Pkc1 pathway die
under all the conditions tested that drive cells into quiescent or
quiescence-like states and that inhibit the TORs. We infer that
the Pkc1p pathway is inherently required for the formation of
viable quiescent cells and not simply for the formation of
quiescent cells that can survive starvation.
Protein Kinase A Pathway
The cyclic AMP (cAMP)-dependent protein kinase (PKA)
pathway is conserved in all eukaryotic cells and, although the
structure of the pathway is not identical in all cells, this path-
way is invariably involved in regulating cell growth and devel-
opment (37, 162). When cAMP concentrations are low, PKA is
inactive and exists as a tetramer composed of two catalytic
subunits and two regulatory subunits (81, 148). There are three
forms of the catalytic subunit encoded by the three partially
redundant genes, TPK1, TPK2, and TPK3 (152). The regula-
tory subunit is encoded by BCY1 (17, 151). When cAMP con-
centrations are high, the nucleotide binds to the inhibitory
Bcy1p subunits, causing dissociation from and activation of the
catalytic subunits (148). Tpk1p, Tpk2p, and Tpk3p appear to
have different functions. For example, cells lacking Tpk2p
192 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

grow better than wild-type cells on nonfermentable carbon
sources, while Tpk1p is actually required for growth on non-
fermentable carbon sources (129), possibly because these cells
arrest prematurely on such nonfermentable carbon sources.
The PKA pathway acts, in general, as an inhibitor of entry
into quiescence. Mutants lacking adenylate cyclase activity are
unable to proliferate and arrest in a state superficially similar
to quiescence (11, 15, 155, 158). Constitutive activation of
PKA, e.g., by deletion of BCY1, causes cell death at the diauxic
shift (16, 17, 166), indicating that proper downregulation of the
PKA pathway is necessary for successful transit to the post-
diauxic phase (96, 140, 157). Altered Bcy1p protein, in which
the serine 145 residue had been changed to alanine, has a
10-fold-higher affinity than does the wild-type protein for the
catalytic subunits. Cultures of cells carrying this allele transit
the diauxic shift and enter stationary phase at a lower cell
density than do wild-type cells (169). In addition, cells harbor-
ing different alleles of bcy1 with mutations in the C terminus
die at different times during the post-diauxic and stationary
phases when cultured to saturation (110). Localization of
Bcy1p and the holoenzyme is dynamic during entry into qui-
escence (54), switching from nuclear localization in exponen-
tially growing cells to cytoplasmic localization as the cells ap-
proach and enter the quiescent state.
If inactivation of the PKA pathway is critical for entry into
quiescence, then activation of the pathway should be important
for successful exit from quiescence when nutrients are again
available. This seems to be the case. Quiescent mutant cells
containing low constitutive activity of the PKA pathway (i.e.,
harboring Tpk-wimpy alleles) display a long delay in reentering
the cell cycle on addition of glucose-based rich medium (70).
How does nutrient availability regulate the PKA pathway?
The immediate upstream regulators of cAMP synthesis are
known. The partially redundant G proteins Ras1 and Ras2 are
activated by signals from the environment, e.g., nutrient avail-
ability. Cdc25p, an exchange factor (130), activates Ras1p and
Ras2p by promoting the replacement of bound GDP to GTP.
The activated (i.e., GTP-bound) forms of these small G pro-
teins directly bind to and activate adenylate cyclase (Cdc35/
Cyr1p) (153), leading to an increase in the level of intracellular
cAMP (for a review, see reference 150).
One G-protein-coupled receptor system (Gpr1p-Gpa2p) ap-
pears to act upstream of PKA as a sensor of external glucose
(46) and is important for glucose activation of cAMP synthesis
(150). However, mutants lacking Gpr1p or Gpa2p are viable
and proliferate normally in glucose-containing media, indicat-
ing that this sensing system plays a minor or specialized role in
the regulation of the PKA pathway. The key regulators of the
pathway during entry into quiescence remain elusive. Although
many observations point to glucose and other carbon sources
as being sensed by the pathway, it has recently been reported
that starvation for nutrients other than carbon can also result
in decreased PKA activity (149).
What are the downstream targets of the PKA pathway? The
pathway inhibits the transcription factors Msn2p and Msn4p,
which are also targets of the TOR pathway (see above) (4,
142). Rim15p, a protein kinase previously shown to stimulate
meiotic gene expression, acts downstream of and is negatively
regulated by PKA (127). Additionally, Gis1p, a putative zinc
finger protein, acts downstream of Rim15p and mediates tran-
scriptional activation via the post-diauxic shift element found
upstream of many genes whose expression increases at the
diauxic shift (111). It has recently been suggested that the PKA
pathway also regulates the Ccr4p-Not complex, which appears
to regulate gene expression both positively and negatively via
the general transcription factor TFIID (85). This Ccr4p-Not
complex may mediate the repression of Msn2p and Msn4p by
the PKA pathway. The PKA pathway may also alter chromatin
structure (184). Finally, the PKA pathway has recently been
shown to be a direct activator of pyruvate kinase (Cdc19p)
(126) and Cox6p (176) in proliferating cells, suggesting a pos-
sible role for the pathway in regulating carbohydrate metabo-
lism and mitochondrial function at the diauxic shift. The spe-
cific role for PKA in the post-diauxic and quiescent phases is
not known, although it may in part regulate the Rye proteins,
several of which are Ssn/Srb subunits of PolII and are required
for survival in stationary phase (reviewed in reference 59).
The Msn2p and Msn4p transcription factors are negatively
regulated by both the TOR and PKA pathways and are acti-
vated at the diauxic shift. Curiously, in strains lacking both
Msn2p and Msn4p, PKA activity is dispensable for vegetative
proliferation (142). Thus, inactivation of the Ras-cAMP path-
way in rich media leads to arrest in a quiescence-like state, in
part because of activation of Msn2p and Msn4p. Importantly,
loss of Msn2p and Msn4p function also confers modest resis-
tance to rapamycin (4). Thus, Msn2p and Msn4p are necessary,
at least in part, for arrest in a quiescence-like state triggered by
PKA or TOR inactivation in the presence of nutrients. How-
ever, activation of Msn2p and Msn4p is not necessary for
successful entry into quiescence triggered by growth of cultures
to stationary phase: msn2 msn4 double mutants have been
reported to maintain viability for protracted periods, although
not as long as do wild-type cells, when starved (95).
Snf1p Pathway
The SNF1 gene encodes the yeast homologue of AMP-acti-
vated protein kinase (AMPK) (56). AMPK is activated by a
variety of stresses to mammalian cells that change the ATP/
AMP ratio, and the activation occurs by direct allosteric
changes (56). Yeast Snf1p is also activated when the in vivo
ATP/AMP ratio drops, but the activation is thought to be
indirect since the purified kinase is refractive to these nucleo-
tides (174). In the presence of glucose, Snf1p is inactive, re-
sulting in the preferential use of glucose as the carbon source
(174, 175). When glucose levels drop, Snf1p is rapidly activated
(within 5 mins) and derepresses the expression of genes re-
quired for the use of alternative carbon sources and metabolic
pathways that generate ATP (174).
Mutants lacking SNF1 cannot utilize alternative carbon
sources such as ethanol and glycerol (56), and they die when
the cultures are grown to high density (actually soon after the
diauxic shift). This and other evidence (see below) suggests
that adaptation to the use of poor carbon sources and the
ability to respire are necessary for proper entry into a stable
quiescent state.
An Emerging Signaling Network
There is accumulating evidence that Snf1p function con-
verges with both the PKA and TOR pathways (and thereby the
VOL. 68, 2004 QUIESCENCE IN YEAST 193

Pkc1 pathway) in modulating various outputs, including Gln3p
and Msn2p/Msn4p (see above) (6, 97). Snf1p activation also
induces peroxisomal genes, such as POT1 (102), which is neg-
atively controlled by PKA (63). In addition to the above inter-
actions with Snf1p, the PKA and Pkc1p pathways appear to
intersect based on studies of Rpi1p, an upstream antagonist of
RAS that also regulates cell wall integrity (143) and the WSC
genes (160). Interactions between the PKA and TOR pathways
are also known to exist through their effects on Msn2p and
Msn4p (39, 159). Thus, the signaling pathways thought to reg-
ulate entry into quiescence appear to form an interacting net-
work that acts at the diauxic shift in response to the change in
carbon quality. The architecture of this putative network is still
poorly defined and may be constant through the life cycle of
yeast or may itself dynamically change as the cells transit into
quiescence.
The link between the TOR and PKA pathways is becoming
clearer. Mutants lacking both Gln3p and Gat2p, known effec-
tors of the Tip41/Tap42 branch of the TOR pathway, are only
moderately resistant to rapamycin. It has been reported that
high- or low-level constitutive activation of the PKA pathway
confers robust rapamycin resistance on such mutants but not
on wild-type cells (134). Further, even in wild-type cells, such
misregulation of the PKA pathway prevents the acquisition of
most, if not all, of the characteristics attributed to regulation of
the Tip41/Tap42-independent branch of the TOR pathway,
including repression of the ribosomal protein genes. Finally, it
has been shown that rapamycin treatment alone causes nuclear
localization of Bcy1, thereby mimicking cAMP depletion and
nitrogen limitation. It thus appears that the Ras-cAMP/PKA
pathway may act, at least in part, downstream of the TORs and
in the Tip41/Tap42-independent branch.
Another recent paper (112) suggests that both pathways
control the activity of the Rim15p protein kinase (128).
Rim15p regulates the expression of genes containing post-
diauxic shift elements in their promoter at or soon after the
diauxic shift. Importantly, rim15⌬ mutants appear to lose via-
bility to some extent when cultured to stationary phase (to 10%
CFU in 30 days), fail to acquire some key characteristics of
quiescence (128), and suppress the growth defects of strains
lacking the PKA pathway activity (as do msn2⌬ msn4⌬ mu-
tants-[see above]).
It now appears that the TOR and PKA pathways have at
least one common target, Rim15p, and regulate multiple com-
mon outputs. It is less clear how this regulation takes place. It
could be that the PKA pathway acts downstream of the TOR
kinases, as suggested by Schmelzle et al. (134). Alternatively,
both pathways may act in parallel, as argued by Pedruzzi et al.
(112). This issue awaits resolution.
Working Model for the Regulation of Entry into Quiescence
Although cells acquire many of the characteristics of quies-
cence at the diauxic shift, it is clear that in rich, glucose-based
medium, entry into quiescence proper occurs when carbon is
finally depleted, concomitant with permanent proliferation ar-
rest. It thus appears, to a first approximation, that growth of a
culture to stationary phase causes at least two distinct changes
in cell state: (i) rapid proliferating (fermenting) to slow pro-
liferating (respiring), concomitant with the diauxic shift of the
culture, and (ii) slow proliferating (respiring) to quiescent,
concomitant with saturation of the culture. The first change (at
the diauxic shift) reprograms cells for respiration, which may
be a necessary precursor for the second change: entry into the
nonproliferating quiescent state (see Fig. 4). Each transition in
liquid medium is triggered by a distinct environmental change:
the first by the lack of a good carbon source, and the second by
the lack of any carbon source (Fig. 4).
The recent work by Gasch et al. (49) has supported this
two-transition model of entry into quiescence after growth of
cultures to stationary phase. One of the experiments in this
work involved monitoring the genome-wide gene expression
changes (by microarray expression profiling) on growth of a
culture for 5 days in rich medium (i.e., to late post-diauxic
shift/early quiescence). Two results are clear. First, the changes
in gene expression that occur at the diauxic shift persist for at
least 5 days in culture. Second, numerous additional changes in
gene expression happen selectively or exclusively after 4 or 5
days, i.e., as the cells approach full quiescence (e.g., induction
of the YDR504w and SNZ1 genes). These late changes in gene
expression are not triggered when cells are starved for nitro-
gen. These changes may thus be specific for entry into quies-
cence proper triggered by carbon starvation.
The putative signaling network involving the TOR, PKA,
PKC, and Snf1p pathways appears to act predominantly at the
first transition, concomitant with the diauxic shift (Fig. 4).
Other, yet to be implicated, pathways may also act here. We
know nothing about the regulators and mediators of the sec-
ond step, final entry into quiescence. Because mutants harbor-
ing different loss-of-function alleles of BCY1, the gene encod-
ing the inhibitor of PKA, appear to lose viability at different
points when cultured to saturation, ranging from the diauxic
shift (reminiscent of a null mutant) to stationary phase (110),
it is possible that stepwise regulation of some or all of the same
signaling network that acts at the diauxic shift also contributes
to the final entry into quiescence.
MAINTENANCE OF VIABILITY IN QUIESCENCE
The processes of entry into and survival in quiescence are
intimately linked. One characteristic of successful entry into
quiescence by wild-type cells must be the acquisition of the
ability to survive in that state. However, success comes in
degrees. A distinction between the processes of entry into and
maintenance in quiescence is possible and useful. Entry can be
viewed as the process by which the key measurable character-
istics of quiescence (yet to be defined) are attained (e.g., in-
volving signaling pathways and mediators of change of state
[see above]); maintenance encompasses the processes by which
the characteristics of quiescence acquired on entry contribute
to long-term viability of that state. Most mutants that lose cell
viability when cultured to stationary phase can in practice be
subclassified as being entry defective or maintenance defective
(see above). In this section, we focus on a few selected pro-
cesses that are thought to be important specifically for main-
tenance of viability in quiescence.
Genes Required for Maintaining Viability
Given the caveat that mutants designated as being required
for “survival” in quiescence/stationary phase can be defective
194 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

in entry into, maintenance in, or exit from quiescence, many
such mutants are likely to be required for maintenance of
viability in quiescence. An increasing number of such mutants
are being identified, and the collection as a whole gives a
low-resolution view of the cellular processes that are more
critical for the survival of quiescent cells than for the survival
of proliferating cells. Mutants known prior to 1993 have been
extensively reviewed already (167). A deficiency in any of a
wide range of cellular functions can cause viability loss in
quiescence; these include oxidative stress responses, e.g., sod2
mutants (45, 90); accumulation of polyphosphate in the vacu-
ole, e.g., ppn1 mutants (136); ubiquitination, e.g., doa4 mutants
(145); and those lacking specific myristolated proteins, e.g.,
arf1, arf2, cdc40/prp17 and las17 mutants (1). Most recently,
genes required for survival in quiescence at 37°C have been
identified among genes coordinately repressed on exiting qui-
escence (171), predominant among which are genes encoding
proteins involved in growth regulation, oxidative phosphoryla-
tion, and other processes involved in mitochondrial function
(M. J. Martinez, A. B. Archuletta, A. I. Rodriguez, A. D. A.
Aragon, S. Roy, C. P. Allen, P. D. Wentzell, and M. Werner-
Washburne, submitted for publication).
“Essential” Genes That Are Not Required for Viability
Another equally interesting set of genes appear to exist:
those that are essential for the proliferative state but are not
required for viability in quiescence. One such example is the
gene encoding the translation factor eIF4E (109). It is thought
that translation initiation in quiescent cells does not involve
recognition of the mRNA cap. Thus, some functions are more
important to a proliferating cell than they are to a quiescent
cell. Although other obvious possibilities could easily be pos-
tulated, e.g., proteins required for cell division cycle progres-
sion should be dispensable during quiescence, we that expect
other, more informative cases will be discovered.
Control of Gene Expression
Correct regulation of gene expression is a key process in the
cell quiescence cycle. A change in the expression of any given
gene can result from altered activity of a particular transcrip-
tion factor (see multiple examples elsewhere in this review) but
can also be affected by changes in general transcription factors.
The latter may also be significant for the cell quiescence cycle.
FIG. 4. Cell transitions that occur when a culture is grown to saturation cell density. As shown in Fig. 1, as a culture of cells is grown to
stationary phase, two distinct and temporally separated transitions occur with concomitant transitions of the constituent cells. At the diauxic shift
transition of the culture, cells switch from fermentation to respiration and from rapid proliferation to slow proliferation. The trigger for this
transition is thought to be exhaustion of a fermentable carbon source. At this transition, the PKA and TOR pathways are downregulated and the
PKC and Snf1 pathways are activated, the former only transiently. At saturation of the culture, cells switch from a slowly proliferating and respiring
state to a quiescent state/G
(0)
that is also thought to be respiring. The trigger for this transition is thought to be depletion of nonfermentable carbon
sources in the medium, i.e., carbon starvation. Mediators of this latter transition are not known, nor has a role for the PKA, TOR, PKA, or Snf1
pathways been established.
V
OL. 68, 2004 QUIESCENCE IN YEAST 195

For example, the general transcription factor TFIID comprises
the TATA box-binding protein and a set of highly conserved
associated factors (TAFIIs). TAFII145, the core subunit of the
yeast TAFII complex, is dispensable for normal transcription
of most yeast genes but is specifically required for progression
through the G
1
/S transition of the cell division cycle. Walker et
al. have shown that the levels of TAFII145, several other
TAFIIs, and TATA box-binding protein are drastically re-
duced in quiescent cells relative to their levels in proliferating
cells (161). Another example is the PolII subunit Rpb4p. Yeast
cells lacking Rpb4p proliferate normally at moderate temper-
atures (18 to 22°C) but not at temperatures outside this range.
When subjected to a heat shock, proliferating cells lacking
RPB4 rapidly lose PolII transcriptional activity and subse-
quently die. When cultured to stationary phase at a permissive
temperature (i.e., permissive for proliferating cells), rpb4⌬ mu-
tants also exhibit a substantial decline in mRNA synthesis
relative to wild-type cells and die. Moreover, in wild-type cells,
the portion of PolII complexes that contain Rrb4p increases
substantially as the cells enter quiescence (21). There is evi-
dence that PolII complexes need to be covalently modified in
some way in order to recruit Rpb4p, and, again, the portion of
modified complexes increases as cells enter quiescence, be-
coming the predominant form (132).
Translation
Protein synthesis consumes a huge amount of the energy in
an exponentially growing yeast cell. rRNA transcription rep-
resents ⬃60% of the total transcription, and ribosomal protein
synthesis represents ⬃15% of total translation. It is not sur-
prising, therefore, that the first coordinated downregulation of
genes that seems to occur during the transition into the qui-
escent state is the coordinated, global shutdown of the tran-
scription of genes coding for the proteins in both subunits of
the ribosome. How this coordinated shutdown is accomplished
is not known. Despite this shutdown of ribosomal protein bio-
synthesis, quiescent cells maintain excess translational capacity
(31) and protein synthesis continues, albeit at very reduced
rates (some 0.3% of the rate in proliferating cells) (47).
A few proteins have so far been identified that are selectively
synthesized after entry into quiescence (47). One such protein,
designated Snz1p, is induced later than all other known pro-
teins, and its relative rate of synthesis increases with time in
quiescence. SNZ1 expression also increases in response to star-
vation for other specific nutrients, such as tryptophan, adenine,
or uracil (106). Increased Snz1p levels may be a hallmark of a
general core quiescence program, one that is shared by the
responses to different starvation regimens and one that is likely
to be highly conserved, as is Snz1p itself (12).
It transpires that Snz1p is required for pyridoxine (vitamin
B
6
) biosynthesis (38, 105). Why, then, is Snz1p (and presum-
ably Snz1p activity) induced in quiescence? There are two
likely possibilities. Vitamin B
6
derivatives may be important
cofactors for metabolism in quiescent cells, possibly in amino-
transferase reactions. Alternatively, these vitamins may func-
tion as antioxidant compounds, providing a defense against
endogenously generated reactive oxygen species, especially sin-
glet oxygen (see below).
Curiously, there is no direct correlation between steady-state
mRNA accumulation and protein synthesis for another pro-
tein, Ssa3p, that is synthesized perferentially in quiescent cells.
It thus appears that the synthesis of at least some important
proteins in the quiescent state is regulated by mechanisms
other than mere control of steady-state mRNA abundance.
Protein Turnover and Covalent Modification of N Termini
Cells in stationary-phase yeast cultures do not increase in
mass with time. Since protein synthesis is known to continue in
these quiescent cells (albeit at a low rate), the obvious conclu-
sion is that protein synthesis and degradation must be tightly
coupled in the quiescent state. However, little is known about
the regulation of protein turnover in quiescent yeast cells.
Evidence from mutants clearly implicates ubiquitin-depen-
dent protein degradation as a process essential for the main-
tenance of viability in quiescent cells. ubi4 mutants, which lack
polyubiquitin (a natural gene fusion of five ubiquitin se-
quences), are unable to maintain viability when cultured to
stationary phase or after starvation for nitrogen or carbon (43,
44). ubc5 and ubc1 mutants, which lack the corresponding
ubiquitin-conjugating enzymes, also display reduced viability in
quiescence (137). In the doa4⌬ mutant, which is defective in
the recycling ubiquitin from ubiquitinated substrates, ubiquitin
is strongly depleted from cells under certain conditions, most
notably as the cultures approach stationary phase (145). Ubiq-
uitin depletion precedes a striking loss of cell viability in sat-
urated cultures of doa4⌬ cells. This loss of viability of doa4⌬
cells is rescued by provision of additional intracellular ubiq-
uitin. Presumably, ubiquitin becomes depleted in the mutant
because it is degraded much more rapidly than in wild-type
cells. Aberrant ubiquitin degradation in the doa4⌬ mutant can
be partially suppressed by mutation of the proteasome or by
inactivation of vacuolar proteolysis or endocytosis. This latter
observation connects protein homeostasis to protein traffick-
ing.
Indirect evidence for the importance of specific targets for
regulated turnover comes from experiments with mutations
that affect N-terminal acetylation. Loss of function of either of
the two subunits of the N-acetyltransferase encoded by the
NAT and ARD1 genes causes a failure of yeast to survive
carbon starvation (107, 172). The proteomes of both wild-type
and nat1 mutant cells in proliferating cultures have been ex-
amined (48). Although only a small subset of the 6,000 yeast
proteins were identified in this analysis, at least 56 proteins
appear to be acetylated by Nat1p under normal proliferative
conditions. Intriguingly, these modified proteins included
Yst1p and Yst2p, structural proteins of the ribosome; Asc1p, a
protein known to interact with the translational machinery;
Ebf1p, a GDP-GTP exchange factor for the translational pro-
tein EF-1; Bmh1p and Bmh2p, proteins known to affect the
PKA and TOR pathways; and Tif1p, translation initiation fac-
tor eIF4A. Others included the ubiquitin-activating enzyme
Uba1p and several peroxisomal proteins. Thus, a number of
proteins involved in functions already implicated in entry into
or maintenance of the quiescent state are targets of N acety-
lation.
Lipidation of the N terminus of some proteins, e.g., by N
myristoylation, is also important for the maintenance of via-
bility in quiescence. S. cerevisiae contains four known acyl co-
196 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

enzyme A (acyl-CoA) synthetases, Faa1p to Faa4p (for “fatty
acid activation proteins”). Acyl-CoA metabolism regulates
protein N myristoylation, a reaction catalyzed by the essential
enzyme, myristoyl-CoA:protein N-myristoyltransferase (Nmt1p).
The combination of a partial-loss-of-function mutation in
NMT1 and a null mutation in FAA4 results in a progressive
millionfold reduction in CFU in quiescence that is associated
with a deficiency in protein N myristoylation (1). This apparent
viability defect first appears during logarithmic growth of cul-
tures, worsens through the post-diauxic phase, and becomes
extreme in stationary phase. Curiously, Nmt1p activity is nor-
mally present in cells cultured to log and diauxic/post-diauxic
phases but is absent from cells at stationary phase. It thus
appears that N-myristoylated proteins present in quiescent
cells, and the requirement for them, are “inherited” from prior
proliferating states.
Many known and putative N-myristoylated proteins have
been identified in yeast (1). Of the 64 genes identified that
encode such proteins, removal of any 1 of the following 9
causes a severe loss of CFU in quiescence: ARF1, ARF2, SIP2,
VAN1, PTC2, YBL049W (homology to SNF7), YJR114W,
YKR007W, and VPS20. Thus, protein N myristoylation (dur-
ing prior proliferating states) and a number of individual tar-
gets of this modification appear to be required for viability in
quiescence.
Autophagy
For turnover of cellular components, eukaryotic cells are
equipped with several other degradation systems, one of which
is the process of autophagy. Autophagy is a membrane trans-
port pathway leading from the cytoplasm to the vacuole in
yeast (or to the lysosomes in mammalian cells) for degradation
and recycling. In addition to nonspecific bulk cytosol, selective
cargoes such as peroxisomes are sorted for autophagic trans-
port under specific physiological conditions. In a nutrient-rich
growth environment, many of the autophagic components are
recruited to execute a specific biosynthetic trafficking process,
the cytoplasm-to-vacuole targeting (Cvt) pathway, that trans-
ports the resident hydrolases aminopeptidase I and ␣-manno-
sidase to the vacuole. Recent studies have identified pathway-
specific components that are necessary to divert a protein
kinase and a lipid kinase complex to regulate the conversion
between the Cvt pathway and autophagy (62).
During the autophagic process, a single-membrane struc-
ture, the so-called isolation membrane, surrounds portions of
the cytoplasm and organelles. Fusion of the tips of the isolation
membrane to each other forms a double-membrane spherical
autophagosome with a diameter of about 1 ␮m. The autopha-
gosome then fuses with lysosomes, and the sequestered con-
tents, along with the inner membranes, are degraded by lyso-
somal hydrolases (100).
In most cells under most conditions, autophagy is usually
suppressed to a very low basal level. Some conditions, includ-
ing starvation (yeast) and hormonal stimulation (mammalian
cells), can trigger dramatic enhancement of autophagy. Auto-
phagy at the basal rate most probably contributes to the turn-
over of cellular components at steady state, whereas starvation-
induced autophagy is thought to aid in maintaining an amino
acid pool for gluconeogenesis and for the synthesis of proteins
essential to survival under starvation conditions.
Autophagy-deficient yeast mutants die rapidly on starvation
(156). Autophagy in yeast has traditionally been stimulated in
rich medium by starving for nitrogen, and the relationship of
this state to the quiescent state attained on starvation for
carbon is unclear. However, increased autophagic activity has
been observed in wild-type cells in cultures entering stationary
phase, and this induction was impaired in a snf1 strain (164).
Snf1p is a putative regulator of entry into quiescence (see
above) and is required for glucose derepression.
Glycogen storage is also defective in autophagy mutants:
mutants defective for autophagy are able to synthesize glyco-
gen when approaching the stationary phase but are unable to
maintain their glycogen stores, because subsequent synthesis is
impaired and degradation by phosphorylase, Gph1p, is en-
hanced. Deletion of GPH1 partially reverses the loss of glyco-
gen accumulation in autophagy mutants. Loss of the vacuolar
glucosidase, SGA1, also protects glycogen stores but does so
only very late in stationary phase, suggesting that Gph1p and
Sga1p may degrade distinct pools of glycogen (164). Defective
glycogen storage in snf1 cells may be due to both defective
synthesis on entry into stationary phase and impaired mainte-
nance of glycogen levels caused by the lack of autophagy,
suggesting an important role for this process in the ability of
cells to survive carbon starvation.
Autophagy may actually help connect transcription and
translation in starved cells. The yeast eIF2␣ kinase, Gcn2
(which is required for translation of the transcription factor
Gcn4p), and the transcription factor Gcn4, which is regulated
by Gcn2, are required for autophagy induced by starvation
(146). This induction process for autophagy is likely to be
functionally conserved since the mammalian eIF2␣ kinase,
PKR, is able to restore starvation-induced autophagy in yeast
cells lacking the GCN2 gene (146).
Interestingly, murine embryonic fibroblasts lacking the
mammalian eIF2␣ kinase or with a nonphosphorylatable mu-
tant form of eIF2␣ (due to a Ser-51 mutation) are defective in
autophagy that can be triggered by herpes simplex virus infec-
tion. Furthermore, PKR and eIF2␣ Ser-51-dependent autoph-
agy is antagonized by the herpes simplex virus neurovirulence
protein, ICP34.5. Thus, autophagy is a novel evolutionarily
conserved function of the eIF2␣ kinase pathway that is both
required for viral virulence and targeted by viral virulence gene
products (146).
Many of the known mediators of entry into quiescence can
modulate autophagy. As noted above, Snf1p is required for
increased autophagy as cultures are grown to saturation (164).
In addition, inhibition of the TOR proteins by rapamycin in
rich media is sufficient to induce autophagy, an induction that
is prevented by hyperactivation of the PKA pathway (134).
Thus, TOR and PKA activities act to repress autophagy in the
vegetative state, and inactivation of these same pathways dur-
ing entry into quiescence probably derepresses autophagy. The
mechanism by which the TOR proteins modulate autophagic
activity is partly understood. The protein kinase activity of
Apg1p, the autophagy-regulating kinase, is enhanced by star-
vation or rapamycin treatment (76). In addition, Apg13p,
which binds to and activates Apg1p, is hyperphosphorylated in
a TOR-dependent manner, reducing its affinity to Apg1p.
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Apg1p-Apg13p association is required for starvation-induced
autophagy but not for the Cvt pathway (74, 76). YPT1, a small
GTPase important for vesicular transport, which is a process
known to be essential for exit from quiescence (see below), has
also been implicated in autophagy via the effects of its GTPase-
activating proteins (Ypt1p-GAPs). Ypt1p-GAP deletion
strains exhibit various morphological alterations resembling
constitutive activation of autophagy (27).
Metabolism
Yeast cells in quiescence have increased amounts of storage
carbohydrates (glycogen and trehalose), whose levels decrease
slowly with time in quiescence. Are these carbohydrates me-
tabolized for fuel? Probably not. The long-term viability of
cells in stationary-phase cultures does not always correlate with
trehalose or glycogen accumulation (141). The primary func-
tion of trehalose may be to protect proteins in quiescent cells
from denaturation and damage by oxygen radicals (5).
There is no answer at present to the obvious question: what
are quiescent cells using as an energy source? The highest
energy output per weight of material in cellular metabolism
comes from the ␤-oxidation of fatty acids. It seems likely,
therefore, that cells in stationary-phase cultures derive their
energy from the slow metabolism of lipids, but no direct evi-
dence for this has been published.
There is indirect evidence to suggest that lipid metabolism is
important in quiescent cells. Loss-of-function mutations in
OPI3, the gene coding for the enzyme that catalyzes the final
methylation reaction in phosphatidylcholine biosynthesis,
cause cells to lose viability when cultured to stationary phase
(98). It is known that as cells enter the quiescent state, triac-
ylglycerol synthesis increases (61), even though total phospho-
lipid biosynthesis decreases (60). These results suggest that
oxidation of triacylglycerols may be an energy reserve for qui-
escent cells.
Since ␤-oxidation of fatty acids occurs in the peroxisomes of
eukaryotic cells, it seems reasonable to assume that there is a
role for the peroxisome in survival of quiescence, but no such
studies on this organelle have been published. Our preliminary
observations (J. L. Collins and G. A. Petsko, unpublished data)
indicate that peroxisomal fatty acid metabolism is not impor-
tant for the maintenance of viability in quiescence. Most other
fatty acid metabolism occurs in mitochondria. Are these or-
ganelles important to quiescent cells? Little work has been
done to answer this question, but there are already good rea-
sons to think that mitochondrial oxidative metabolism may be
the chief source of energy for quiescent cells. Glyoxalate path-
way genes are upregulated in cell cultures on entry into sta-
tionary phase (49). More directly, petite mutants and mutants
harboring other mitochondrial loss-of-function defects die rap-
idly when starved for carbon in rich media (J. L. Collins, G. A.
Petsko and D. Ringe, unpublished data; Martinez et al., sub-
mitted).
Redox Homeostasis
Unlike proliferative cells, quiescent yeast cells cannot dilute
out damage to proteins and DNA by rapid synthesis of new
macromolecules and cell division. Hence, quiescent cells are
potentially more vulnerable to internal and external stresses
than are proliferating cells. It is reasonable to assume that
quiescent cells have active, maybe even specialized, protection
mechanisms to counter any accumulating damage. Here, we
focus on one such stress, oxidative damage.
Mitochondrial respiration appears to be a major source of
energy for quiescent cells. Unfortunately, respiration produces
large amounts of reactive oxygen species, whose toxic effects
must be countered if viability is to be maintained. We think
that proper redox homeostasis is of great importance to qui-
escent cell viability. Multiple findings support this view. For
example, the expression of genes encoding antioxidant en-
zymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide
dismutase (Cu,ZnSOD), and glutathione reductase, is induced
when quiescent cells are exposed to menadione, an oxidizing
agent (26). Thus, quiescent cells retain a capacity to detect and
respond to oxidative damage.
It is clear that the response to oxidative stress is important.
Longo et al. (92) studied yeast mutants lacking CuZnSOD and
MnSOD (sod1 and sod2, respectively) and determined their
long-term viability (by measuring CFU) in stationary-phase
cultures in minimal medium. Such cells would be in a state
related to, but not identical to, our reference quiescent state.
In well-aerated cultures, the lack of either SOD resulted in
dramatic loss of viability over the first few weeks in culture.
However, the double mutant died more quickly still, i.e., within
a few days. Reduction of respiration via a second mutation
dramatically increased short-term survival. These results
strongly suggest that ongoing mitochondrial respiration is itself
a major stress to starved yeast cells.
Aging versus Maintenance of Viability
The measurable, time-dependent loss of CFU in stationary-
phase yeast cultures in synthetic media has been proposed as a
model for cellular aging, “chronological” ageing (41). Station-
ary-phase cultures in rich media are much more resilient to
apparent loss of viability than are saturated cultures in syn-
thetic media for reasons that are not yet understood. Never-
theless, the processes in cells grown to saturation in synthetic
medium should, in large part, inform us of the evolution of our
reference quiescent state, and vice versa.
Another type of aging in yeast is replicative aging, defined as
the loss of potential to undergo subsequent rounds of cell
division cycle in rich media. A newly born daughter cell can
undergo only a finite number of subsequent cell divisions be-
fore becoming senescent: mutants with a longer replicative life
span can undergo more rounds of division from birth to senes-
cence in the continuous presence of ample food. It is not clear
how the two aging processes, chronological and replicative, are
related: some mutation appear to have opposite effects on
them, lengthening one while shortening the other, whereas
other mutants affect one process only. However, there are a
few genes whose deletion appears to affect both ageing mech-
anisms in the same way, suggesting that there is some com-
monality between the two mechanisms. For example, deletion
of SCH9, a gene encoding a protein kinase that is a possible
yeast homologue to the human antiapoptotic kinase Akt/PKB,
dramatically extends the chronological life span of yeast (42) in
a SOD2-dependent manner (40).
198 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

There is also evidence from yeast studies that longevity in
eukaryotes may be negatively regulated by the PKA pathway,
which is implicated in entry into quiescence. Mutations that
decrease the activity of the Ras-Cyr1p-PKA pathway extend
the longevity of yeast cells and increase stress resistance by
activating transcription factors Msn2 and Msn4 and the mito-
chondrial superoxide dismutase Sod2p. Specifically, deletion of
RAS2, one of the two Ras genes in yeast, doubles the chrono-
logical life span (overexpression of RAS2 also increases the life
span, suggesting that the dependence of life span on Ras2p
activity is denoted by a bell-shaped curve); transposon muta-
tion of CYR1, the adenylate cyclase that is a downstream target
of Ras2p, has a similar effect (40). We presume that extension
of life span, as opposed to its shortening, cannot be due to a
failure to enter quiescence proper, so these observations sug-
gest a role for the Ras-Cyr1p-PKA pathway in maintenance as
well as entry and also suggest a possible connection between
these stages of the quiescence cycle.
It is known that cells lose replicative capacity with time spent
in quiescence proper, demonstrating a direct relationship be-
tween replicative ageing and evolution of quiescence. It must
be noted, however, that the very concept of yeast as a model
for aging in other organisms has been questioned (50). The
specific objections raised do not invalidate the relationships
between replicative ageing, chronological ageing, and mainte-
nance of viability in quiescence in yeast; the study of one
should, in part at least, inform the others.
EXITING FROM QUIESCENCE
Resuspending quiescent cells in media containing all neces-
sary nutrients (including a carbon source) stimulates exit from
quiescence and completion of the quiescence cycle. Stimulated
(refed) quiescent cells lose thermotolerance, become sensitive
to cell wall-degrading enzymes, and display increased rates of
RNA and protein synthesis. Internal carbohydrate stores such
as glycogen and trehalose are also mobilized. Ultimately, stim-
ulated cells resume cell growth and begin proliferation, i.e.,
enter the proliferative cell cycle (167).
Sensing Nutrients
How does a quiescent cell sense the presence of nutrients? It
is possible that the cell has the ability to simultaneously sense
the presence of all essential nutrients such that it initiates exit
from quiescence only in complete medium. Alternatively, and
more economically, a quiescent cell may be poised to detect
only one or a few key nutrients that would indicate, with
sufficiently low risk, that the environment has become permis-
sive again for active proliferation.
Quiescent yeast cells appear to be “risk takers” and primar-
ily use the presence of an external carbon source as the key
indicator of a favorable change in nutritional fortune. Cells
allowed to enter quiescence in glucose-based rich medium are
able to maintain viability for long periods even when subse-
quently transferred to distilled water (51, 52). Nevertheless,
the simple addition of a carbon source such as glucose to
quiescent cells in water causes the loss of many characteristics
of quiescence (52).
This striking finding indicates that the presence of a carbon
source alone is sufficient to initiate, at least in part, the process
of exiting from quiescence. In contrast, quiescent cells in water
are refractile to the addition of a nitrogen source alone, sup-
porting the high predictive value placed by these cells on car-
bon availability. This predictive gamble can be risky: the addi-
tion of glucose to quiescent cells in water ultimately results in
cell death, presumably due to the lack of other essential nu-
trients. The presence of a carbon source alone appears, there-
fore, to irreversibly commit quiescent cells to attempting to exit
from quiescence.
Transcriptional Changes
An analysis of genome-wide gene expression changes that
occur during exit from quiescence has been performed using
slide-based microarrays and at 5-min intervals after stimulation
(Martinez et al., submitted). A correlation map of this time
course revealed that the greatest changes in gene expression
occur within the first 10 to 15 min of stimulation by addition of
nutrients. For example, at least 127 genes, including SNZ1, are
rapidly and coordinately repressed. In addition, the “ribo-
somal” gene set encoding ribosomal proteins and related trans-
lation factors (and a potential target of the PKA and TOR
pathways [see “Entry into quiescence” above]) is coordinately
induced within the first 10 min. Over the extended time course,
expression of approximately one-third of all genes is altered by
a factor of 2 or greater. Distinct temporal patterns of expres-
sion are observed, indicating that exiting from quiescence is an
ordered set of sequential events.
It has long been thought that stimulated cells exit from
quiescence into the G
1
phase of the cell cycle. Ultimately this
may be the case, and some genes characteristically expressed in
G
1
, such as SW14, are rapidly induced upon refeeding. How-
ever, exiting cells appear to transit through a unique set of
states that do not appear to be similar to any other known state
of yeast. The expression profiles of exiting cells appear to be
distinct from that of cells in the G
1
phase of the cell cycle or
cells at any intermediate stage during entry into quiescence
(M. J. Martinez and M. Werner-Washburne, unpublished
data). This result requires further study to distinguish the phys-
iological from the cell cycle responses during the exit process
and to ensure that the cells being studied are synchronous.
Nevertheless, this surprising finding suggests that cells exiting
from quiescence take a unique path back to the proliferating
state, a path that is not simply the reverse of that taken during
entry into quiescence.
The rapid transcriptional response of a quiescent cell to the
addition of nutrients leads to additional questions. What is the
source of nucleoside triphosphates for this synthesis, and
where are they stored? It is possible that early transcription
utilizes internal stockpiles retained during quiescence. How is
a quiescent cell poised to make such an enormous and coor-
dinated response? Such a rapid response doubtless requires
activation or remodeling of signal transduction pathways, chro-
matin structure, transcription factors, and RNA polymerases.
We do not know the answers to these questions.
In another study, Brejning et al. (13, 14) examined global
changes in gene expression after resuspending cells that were
post-diauxic (but not yet quiescent) in synthetic defined me-
dium containing all essential nutrients. They focused on the lag
VOL. 68, 2004 QUIESCENCE IN YEAST 199

phase, i.e., the time between stimulation by addition of nutri-
ent and the actual resumption of rapid cell proliferation. Ap-
proximately 240 genes were induced, and 122 genes were re-
pressed at least fivefold during the lag phase. Again, the
expression profiles indicate that lag-phase cells display expres-
sion patterns that are distinct from those of actively prolifer-
ating or post-diauxic cells.
Proteome Changes
No systematic analysis of changes in the proteome during
exit from quiescence has been reported to date. However, the
proteome has been monitored by two-dimensional gel electro-
phoresis during the lag phase on resuspension of post-diauxic
shift cells in fresh synthetic complete medium (13). The overall
rate of protein synthesis increased dramatically during the lag
phase, with a concomitant increase in the number of proteins
detectable on a single two-dimensional gel, from approxi-
mately 500 in early lag phase to 1,500 in late lag phase. The
increased abundance of a protein correlated well with an in-
creased amount of the corresponding transcript. Thus, there is
little evidence to date for wholesale posttranslational control
of protein abundance during the lag phase. These results may
be relevant to the process of exiting from quiescence.
Exit Mutants
The study of mutants defective in exiting from quiescence
should complement studies of mutants defective in entry. An
analogy can be drawn to the response of haploid cells to the
presence of mating pheromone. The addition of mating pher-
omone to haploid yeast cells causes a change in cell state from
the proliferative state to the nonproliferative shmoo. Subse-
quent removal of pheromone reverses this change in state.
Mutants lacking activators of the mating-signal transduction
pathway, such as components of a MAP kinase cascade, fail to
respond to pheromone treatment, i.e., cannot enter the shmoo
state (144). In contrast, mutants lacking inhibitors of the mat-
ing pathway, e.g., protein phosphatases that inactivate the
MAP kinase cascade, are defective in resuming proliferation
after pheromone removal, i.e., cannot exit from the shmoo
state (18, 19). Similarly, mutants unable to exit from quies-
cence should be defective in genes encoding distinct and op-
posing regulators and mediators to those identified from ge-
netic analysis of entry.
The process of exit from quiescence has received little at-
tention from geneticists to date, although, as outlined above,
many such mutants may have been inappropriately classified.
Only one mutant, gcs1, has been confirmed to be specifically
defective in exiting from the quiescent state (34, 35, 59, 167).
Gcs1 and Vesicular Traffic
Mutants lacking the GCS1 gene are conditionally (at low
temperature, e.g., 15°C) unable to successfully exit from qui-
escence on stimulation by fresh nutrients (34, 35, 59, 64, 167).
Because null alleles of GCS1 display this phenotype, the con-
ditionality is due to a conditional requirement for Gcs1p ac-
tivity. The defect is specific for exit from quiescence, since gcs1
mutants do not display any problems in other phases of the life
cycle, although diploid cells lacking Gcs1p function are im-
paired for sporulation (34, 35, 71, 72) (G. C. Johnston and
R. A. Singer, unpublished data). Quiescent gcs1 mutants are
viable (see below) and can successfully return to active cell
proliferation at temperatures higher than 15°C. The special
requirement for Gcs1p during exit from quiescence at low
temperatures is imposed relatively early in the process of en-
tering into quiescence, since gcs1 cells become conditionally
unable to resume proliferation soon after the diauxic shift (35,
64, 71, 72). As indicated above, this degree of starvation is also
sufficient to cause cells to acquire many of the hallmarks of
quiescence (for reviews, see references 72 and 167).
At 15°C, the resupply of nutrients to starved gcs1 cells stim-
ulates cell growth (i.e., mass and volume increase), RNA and
protein synthesis, degradation of storage carbohydrates, and
gene expression changes characteristic of exiting cells (35).
Indeed, resupply of nutrients causes the appearance of mRNA
transcripts from the GCS1 gene itself at a time when other
transcripts also become detectable (64). The kinetics of these
responses is similar to that seen for wild-type cells exiting from
quiescence under the same conditions. However, gcs1 mutant
cells subsequently fail to reenter the mitotic cell cycle and pass
the START checkpoint (34, 35, 64). This behavior suggests
that the gcs1 mutation does not affect the ability of a quiescent
cell to sense and initially respond to the presence of nutrients
but, rather, impairs some later process required to fully achieve
the proliferative state.
The role of Gcs1p in the late quiescence cycle is suggested by
the nature of the Gcs1p protein itself (64). Gcs1p is a GTPase-
activating protein (GAP) that stimulates GTP hydrolysis by the
Arf small GTP-binding proteins (118). Arf proteins are known
to regulate various stages of vesicular transport in proliferating
cells (reviewed in references 9 and 33), suggesting that the
remodeling of intracellular vesicular transport may be critical
for the transition from the quiescent to the proliferating state.
GCS1 expression is not restricted to, and is not uniquely
affected by, exit from quiescence. Thus, Gcs1p probably plays
a role both in the vegetative state and in exiting from the
quiescent state. These roles may be the same or distinct.
What is the role of Gcs1p in proliferating cells? The yeast
genome encodes several proteins that are related in structure
and function to Gcs1 (115, 116). One of these, Age2p, is an-
other ArfGAP whose in vivo function in proliferating cells
overlaps that of Gcs1p: although gcs1 or age1 single mutants
cells proliferate normally, the gcs1 age1 double mutant is invi-
able (117, 182). This overlapping and essential function of the
two proteins in proliferating cells appears to be to enable
transport of vesicles from the trans- Golgi network (117). The
double mutant lacking both proteins displays a severe impair-
ment in endosomal vesicle traffic.
What is the role of Gcs1p during exit from quiescence? gcs1
mutants undergoing the transition from quiescence back to cell
proliferation at a low (restrictive) temperature also display a
severe endosomal impairment (163), similar to that shown by
vegetative cells lacking both Gcs1p and Age1p (117). Although
it has not yet been demonstrated conclusively, this vesicle traf-
ficking defect may account for the failure of gcs1 mutants to
successfully exit from quiescence. In support of this possibility,
replacing the in vivo wild-type allele of GCS1 with a mutated
version encoding a GAP-dead form of the protein (178) mim-
200 GRAY ET AL. MICROBIOL.MOL.BIOL.REV.

ics the exit defect of gcs1 null mutants (C. L. Adams, S. Lewis,
R. A. Singer, and G. C. Johnston, unpublished data).
If Gcs1p performs the same function in cells exiting from
quiescence as it does in proliferating cells, why do gcs1 single
mutants display a specific defect in transiting from the quies-
cent to the proliferating state at low temperature? There are
three likely possibilities. First, a cell exiting from quiescence
may require an unusual amount of endosomal trafficking to
successfully and physically transform itself into a proliferation-
competent state, especially at low temperature. However, re-
duction of the overall ArfGAP activity of a cell by deletion of
the AGE2 gene alone does not affect its ability to exit from
quiescence at any temperature, making this possibility unlikely.
Second, it may be that Age2p function (or some component of
an Age2p-mediated pathway) is impaired during exit from
quiescence, particularly at low temperatures. In this scenario, a
stimulated gcs1 mutant cell would effectively lack both Age2p
(or Age2p-related) and Gcs1p functions at low temperature,
only the latter because of mutation. Finally, Gcs1 may perform
another function that may directly or indirectly affect endoso-
mal traffic and that is not shared with Age2p. Such a function
could be unique to, or uniquely required for, exit from quies-
cence at low temperatures. A possible function has recently
come to light. A subset of mammalian ArfGAP proteins, the
centaurins (125), regulate both vesicle transport and rear-
rangements of the actin cytoskeleton at the plasma membrane
and endosomal compartments. Gcs1p appears to be the yeast
homologue of centaurin-␣ (8). Interestingly, gcs1 mutants dis-
play abnormal actin cytoskeletal organization specifically when
exiting from quiescence at 15°C (Johnston and Singer, unpub-
lished). Thus, a defect in actin organization may underlie, at
least in part, the defect in resumption of cell proliferation from
quiescence (28) exhibited by gcs1 mutants.
CONCLUSIONS AND PERSPECTIVES
Quiescence is poorly understood for any organism. Even for
yeast, progress has been limited. There are many possible
reasons for this, as discussed above, including the apparent
modest life-style of quiescent cells and misconceptions about
their viability. However, additional factors appear to have
clouded the field.
Herein, we focus attention on only one quiescent state of the
cell, i.e., that achieved by growth of liquid cultures to satura-
tion in rich medium. We currently call this state “quiescence”
and limit the use of the term to this case alone. This is an
operational definition only. It allows us relate the findings of
different researchers who have worked on cells derived by the
same operation. In reality, it is likely that yeast cells can enter
any one of a series of related and stable quiescence-like states
depending on the environmental trigger. For example, nonpro-
liferating states can be triggered by starvation for carbon, ni-
trogen, or phosphate in rich or synthetic defined media, by
sporulation of diploids, and even by transfer of proliferating
cells to distilled water. We do not know if the states entered as
a result of all these treatments are largely similar (i.e., if there
is a core quiescence program) or not. Because of this uncer-
tainty, data acquired for any one state may or may not be
relevant to any of the others.
For rapid progress to be possible in the study of quiescent
states, it is critical that each state be studied separately. This
has not been the case to date. For example, as argued above,
cells in cultures that have just arrested at or passed the diauxic
shift are not in the same state as cells in saturated cultures, yet
the two cell states have often been confused. Cells from sta-
tionary-phase cultures (i.e., cells starved for carbon) have been
studied more intensively than have other nonproliferating cells
and as such are the most understandable.
However, focusing attention on one state for study does not
necessarily help with gleaning information and understanding
from cells that do not appear to do much. Thankfully, recent
advances in experimental methodologies and increasing avail-
ability of reagents derived from the exploitation of the genome
sequence are coming to the rescue.
A first step toward fully understanding the biology of quies-
cent cells is to characterize the quiescent state itself and the
way in which it differs from the proliferating state. For exam-
ple, genome-wide gene expression profiling (see, e.g., refer-
ence 177) can be and is being done to uncover the differences
between the gene expression patterns of quiescent and prolif-
erating cells (49, 171). Furthermore, large-scale analysis of the
proteome is increasingly viable, including assays of the protein
concentration, localization, covalent modification, and com-
plex formation (see, e.g., references 165 and 183).
Thus, in addition to the known measurable phenotypes of
quiescent cells, such new technologies will add countless oth-
ers. The definition of the quiescent state would allow a com-
parison with other nonproliferating states, permitting the re-
lationships between all of them to be finally estimated with
good confidence. Furthermore, knowledge of the unique prop-
erties of quiescent cells will contribute to our understanding of
the processes by which these cells remain so stubbornly viable.
Studying the dynamic processes of entry into and exit from
quiescence in similar detail should be particularly informative.
Which signaling pathways regulate each stage during entry and
exit? This question is potentially answerable. Furthermore, it
should not be assumed that maintaining viability in quiescence
is not itself dynamic. It is likely that the state of the cell evolves
with time in quiescence, as internal energy reserves become
depleted and as the cell copes with ongoing environmental
assault, e.g., oxidative stress. We know that this dynamic evo-
lution of the quiescent state is likely to occur, because a related
phenomenon in yeast is well known, i.e., chronological aging
(see above).
Understanding how a cell physically transits back and forth
between the proliferating and quiescent states is a more for-
midable challenge, since such transitions appear to involve the
wholesale remodeling of many (if not most) cell processes and
structures. Even a partial list of the known and coordinated
changes associated with these transitions includes changes in
many signal transduction pathways; chromatin structure; tran-
scription rate and pattern; mRNA stability; translation rate,
pattern, and mechanism; protein stability; covalent modifica-
tion of protein; vesicular traffic; mitochondrial structure; and
cell wall structure. Beneath this complexity lies a big reward:
understanding the mechanisms by which the many cellular
processes that underlie active growth, and that are mostly
studied in isolation in proliferating cells, are coordinately reg-
ulated.
Analytical methods alone will be very useful, but the com-
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bination of genetics and these technologies promises to gen-
erate the main revolution in our understanding of quiescence.
As outlined above, the subclassification of mutants that lose
colony-forming potential after growth to stationary phase
means that mutants defective in individual aspects of entry
into, maintenance in, and exit from quiescence can be identi-
fied. The availability of genome-wide deletion collections and
the ability to screen such collections robotically means that
large numbers of cell quiescence cycle (cqc) mutants will soon
be identified.
One of the motivations for studying quiescence in yeast is
the hope that it might prove to be a model for the behavior of
quiescent mammalian cells (93). A number of cells in more
complex eukaryotes exit the mitotic cell cycle permanently on
terminal differentiation. The most important in terms of hu-
man health and disease are the neurons (181). Humans are
prey to a host of neurodegenerative diseases, both sporadic
and inherited. Most of these diseases seem to result from the
accumulation in neurons of aggregates of misfolded proteins
(139), although the exact mechanism of neurotoxicity is not
established. Increased oxidative stress has been implicated in
the etiology of many of these diseases: for example, oxidatively
damaged proteins are present in the aggregates found in neu-
rons of patients with Alzheimer’s disease and Parkinson’s dis-
ease (133), and loss-of-function mutations in DJ-1, a protein
implicated in the response of the cell to oxidative stress, cause
early-onset Parkinson’s disease (10, 173, 180). In the familial
forms of many neurodegenerative diseases, the mutated pro-
teins are expressed in many different cell types (DJ-1, for
example, is ubiquitous), yet the predominant phenotype is the
death of specific classes of neurons.
What makes neurons particularly vulnerable to protein dam-
age and/or the loss of proteins that protect against such dam-
age? It is tempting to conclude that at least part of the expla-
nation is that these are quiescent cells (albeit ones that are
highly active in certain specific metabolic processes) and there-
fore cannot dilute out or repair the damage as efficiently as
proliferating cells can (139). Understanding the factors respon-
sible for the survival—and death—of quiescent yeast cells,
which have homologues for many of the genes encoding pro-
teins such as DJ-1 and SOD, associated with human disease,
may lead to a better understanding of the vulnerability of
neurons to degeneration (122) and, it is hoped, also give clues
to how such degeneration may be prevented.
Recently, evidence has also emerged that degenerating neu-
rons in several different human diseases display markers indi-
cating that they have attempted to exit from G
(0)
/quiescence
and reenter the cell cycle, including expression of cyclin-de-
pendent protein kinases and their regulators (55). Reexpres-
sion or deregulation of the genes involved in exit from quies-
cence may thus be an important step in neurodegeneration. A
better understanding of this stage of the cell quiescence cycle
may present new opportunities for therapy (101, 123, 179).
In conclusion, we hope that it has become clear that the cell
quiescence cycle is as important a process to life on this planet
as is the mitotic cell division cycle. By the time of our next
review, we hope that the cell quiescence cycle is as active and
productive a topic of study as is the cell division cycle today.
ACKNOWLEDGMENTS
We thank the members of the Gray, Werner-Washburne, Petsko/
Ringe, and Johnston/Singer laboratories for helpful discussions. One
of us (G.A.P.) is especially grateful to Jeremy Thorner, who first
brought the phenomenon of stationary-phase survival to his attention
and who encouraged his budding interest (pun intended).
Work in the Gray laboratory has been supported by funds from The
Royal Society, the Wellcome Trust, the IBLS Research Committee,
and the Biotechnology and Biological Sciences Research Council. The
Petsko and Ringe laboratories thank the Ellison Medical Foundation
for support. Research in the Johnston/Singer laboratory has been
supported by funds from the Canadian Cancer Society through grants
from the National Cancer Institute of Canada and grants from the
Canadian Institutes for Health Research. Research in the Werner-
Washburne laboratory has been supported by funds from the National
Institutes of Health.
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