Sucrose Utilization in Budding Yeast as a Model for the
Origin of Undifferentiated Multicellularity
John H. Koschwanez1,2*, Kevin R. Foster1,3,4, Andrew W. Murray1,2
1FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America, 2Department of Molecular and Cellular Biology, Harvard
University, Cambridge, Massachusetts, United States of America, 3Department of Zoology, University of Oxford, Oxford, United Kingdom, 4Oxford Center for Integrative
Systems Biology, University of Oxford, Oxford, United Kingdom
We use the budding yeast, Saccharomyces cerevisiae, to investigate one model for the initial emergence of multicellularity:
the formation of multicellular aggregates as a result of incomplete cell separation. We combine simulations with
experiments to show how the use of secreted public goods favors the formation of multicellular aggregates. Yeast cells can
cooperate by secreting invertase, an enzyme that digests sucrose into monosaccharides, and many wild isolates are
multicellular because cell walls remain attached to each other after the cells divide. We manipulate invertase secretion and
cell attachment, and show that multicellular clumps have two advantages over single cells: they grow under conditions
where single cells cannot and they compete better against cheaters, cells that do not make invertase. We propose that the
prior use of public goods led to selection for the incomplete cell separation that first produced multicellularity.
Citation: Koschwanez JH, Foster KR, Murray AW (2011) Sucrose Utilization in Budding Yeast as a Model for the Origin of Undifferentiated Multicellularity. PLoS
Biol 9(8): e1001122. doi:10.1371/journal.pbio.1001122
Academic Editor: Laurent Keller, University of Lausanne, Switzerland
Received March 19, 2011; Accepted June 29, 2011; Published August 9, 2011
Copyright: ? 2011 Koschwanez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: JK is supported by NIH NIGMS award K25GM085806, KF is supported by European Research Council Grant 242670, and AM is supported by NIGMS
Center for Systems Biology (GM068763). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: FACS, fluorescence activated cell sorter; OD, optical density; PMT, photomultiplier tube; YNB, yeast nitrogen base.
* E-mail: email@example.com
During evolution, smaller and simpler elements have repeatedly
come together to make bigger and more complicated functional
units; examples include genes forming genomes and individuals
forming societies. Multicellular organisms are societies of cells and
the transition from single to multicelled groups arises in two ways
[1,2]: (1) single cells come together to form groups that
subsequently differentiate into different cell types (e.g., slime
molds and myxobacteria), or (2) the offspring of a single cell stay
stuck together after cell division. This second mode—incomplete
cell separation—appears to be a critical step in the independent
origins of multicellularity that led to animals, plants, and colonial
algae . However, the origins of incomplete cell separation are
obscure: the ancestors of current multicellular organisms are
ancient and the interpretation of early multicellular fossils [4,5]
remains a challenge .
Despite these difficulties, taxonomic groups that contain both
multicellular and unicellular species have provided insights into
the origin of multicellularity. The Volvocaceae are a family of
algae that range from single celled species through undifferentiated
groups of cells to species with differentiated germ line and somatic
cells. In this group multicellularity appears to have arisen through
a series of stages with incomplete cell separation occurring early on
in the transition . The choanoflagellates, which are related to
basal animals such as sponges, exist in both single celled and
colonial forms, and also form colonies through incomplete cell
division . We focus on what was likely to be the initial step in
the evolution of multicellularity, the appearance of aggregates of
undifferentiated groups of cells, and ignore two crucial later stages
common to plants and animals: the division of labor between
different cell types and reproduction through single-celled
We used the genetic tractability of the budding yeast,
Saccharomyces cerevisiae, to study the simplest form of multicellularity:
an undifferentiated group of cells that remain attached to each
other after cell division. Our goal was to find conditions where
cells that remain attached to one another have an advantage over
isolated cells. We genetically manipulated two traits of budding
yeast. The first is cell separation. After cytokinesis, the physical
separation of the two daughter cells requires digestion of part of
the cell wall . Many natural isolates of S. cerevisiae show
incomplete separation and form clumps, whereas laboratory
strains have been selected to show complete separation and exist
as isolated cells . The second is the secretion of hydrolytic
enzymes that act on more complex molecules to release nutrients,
which act as public goods that cells can take up. Enzyme secretion
is a form of cooperation because the nutrients the enzymes release
can increase the fitness of cells other than the secreting cell. Yeast
secrete a number of enzymes, including acid phosphatase (Pho5)
, phospholipase (Plb2) , and invertase (Suc2) , that
release nutrients from molecules in the medium. Here we focus on
Invertase breaks down the disaccharide sucrose into the
monosaccharides glucose and fructose. The secretion of invertase
from budding yeast has long been studied. In the 19thcentury,
PLoS Biology | www.plosbiology.org1August 2011 | Volume 9 | Issue 8 | e1001122
Berthelot and Pasteur quarreled over the mechanism responsible
for the invertase action  and Fischer’s studies of invertase in
the early 20thcentury led to the ‘‘lock and key’’ concept of enzyme
specificity . More recently, key aspects of glucose repression
and protein secretion were discovered by studying invertase [16–
22], and invertase secretion has served as a model for studies of
cooperation among budding yeast [23,24].
Here we explore the interaction between incomplete cell
separation and the use of invertase as a secreted product that
promotes the growth of neighboring cells. Our goal was to ask if
cooperative enzyme secretion and the formation of groups of
genetically identical cells could have led to the origin of
multicellular life. Our data suggest that the use of secreted
products can indeed lead to natural selection for incomplete cell
Lab Yeast Cannot Grow from a Single Cell in Low
Concentrations of Sucrose
We began by characterizing the growth of single yeast cells in
medium with sucrose as the only carbon source, an environment
that requires invertase secretion to allow cell proliferation. At low
glucose concentrations, invertase, encoded by the SUC2 gene, is
secreted in a glycosylated, octameric form (Figure S1) [25,26]. The
invertase octamer is retained in the cell wall, where it hydrolyzes
the sucrose in the media into glucose and fructose. After
hydrolysis, each glucose and fructose molecule either diffuses
away from the cell or is captured by sugar transporters in the cell
membrane (Figure 1A). The sugar influx into the cell therefore
depends on the rates of sucrose diffusion to the cell wall, sucrose
hydrolysis at the cell wall, and capture of the diffusing
monosaccharides at the cell membrane. Contrast this with the
case of a cell grown in glucose and fructose, where the sugar flux
into the cell depends only on the rate of monosaccharide diffusion
and capture at the cell membrane. If three conditions are satisfied,
there should be a sugar concentration that allows growth on
glucose and fructose but not on sucrose: (1) the net monosaccha-
ride flux into a cell grown in sucrose is less than the
monosaccharide flux of a cell grown in equivalent molarity
glucose and fructose, (2) there is minimum monosaccharide flux
required for growth, and (3) there is no sucrose import into the
cell. In addition, the threshold concentration for growth on sucrose
should depend on cell density because some of the monosaccha-
rides that escape from one cell can be captured by its neighbors.
To test these predictions, we used a fluorescence activated cell
sorter (FACS) to inoculate between 1 and 512 single budding yeast
cells of a standard laboratory strain background (W303) into each
well of a 96-well microtiter plate. Each well contained 150 ml of
media that contained one of two carbon sources: sucrose or a
mixture of glucose and fructose. The plates were examined after
being left stationary at 30uC for 85 h. Figure 2A shows that each
cell placed into medium containing 4 mM glucose plus 4 mM
fructose formed a visible microcolony, whereas Figure 2B shows
that even at 8 mM sucrose (equivalent to 8 mM glucose plus
8 mM fructose), inoculating as many as 512 single cells per well
failed to lead to visible growth. Growth at 16 mM sucrose was cell
density dependent: very few of the wells inoculated with a single
cell produced visible growth, but there was growth in every well
inoculated with 512 cells. Figures 2B and S2 show that two
different strain backgrounds, W303 and S288C, gave similar
results. (All strains in this study are prototrophic and constitutively
express a fluorescent protein to allow FACS selection and
fluorescence-based imaging.) The results in Figures 2B and S2
cannot be explained by cells making a stochastic decision whether
to proliferate or not in sucrose. For this to be the case, a small
Figure 1. Extracellular hydrolysis of sucrose allows other cells
to share glucose and fructose. (A) Sucrose is hydrolyzed into
glucose and fructose by invertase located in the cell wall. The glucose
and fructose are imported into the cell by hexose transporters or escape
into the medium by diffusion. (B) The glucose and fructose
monosaccharides diffuse away from the cell wall and are more easily
shared between cells when the cells are clustered in a clump (right)
than when the cells are spaced apart (left).
The evolution of multicellularity is one of the major steps
in the history of life and has occurred many times
independently. Despite this, we do not understand how
and why single-celled organisms first joined together to
form multicellular clumps of cells. Here, we show that
clumps of cells can cooperate, using secreted enzymes, to
collect food from the environment. In nature, the budding
yeast Saccharomyces cerevisiae grows as multicellular
clumps and secretes invertase, an enzyme that breaks
down sucrose into smaller sugars (glucose and fructose)
that cells can import. We genetically manipulate both
clumping and secretion to show that multicellular clumps
of cells can grow when sucrose is scarce, whereas single
cells cannot. In addition, we find that clumps of cells have
an advantage when competing against ‘‘cheating’’ cells
that import sugars but do not make invertase. Since the
evolution of secreted enzymes predates the origin of
multicellularity, we argue that the social benefits conferred
by secreted enzymes were the driving force for the
evolution of cell clumps that were the first, primitive form
of multicellular life.
Multicellularity and Sucrose Utilization in Yeast
PLoS Biology | www.plosbiology.org2 August 2011 | Volume 9 | Issue 8 | e1001122
tion of algorithm.
Parameters used for software simulation and descrip-
Yeast nitrogen base recipe.
exponentially growing cells.
Fitness cost of endogenous invertase expression for
The authors thank Joao Xavier for discussions about diffusion and
members of the Murray Lab for reviewing the manuscript.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: JHK KRF
AWM. Performed the experiments: JHK. Analyzed the data: JHK KRF
AWM. Wrote the paper: JHK KRF AWM.
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