T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 181, No. 2, April 21, 2008 281–292
R.A. Saleem and B. Knoblach contributed equally to this paper.
Correspondence to John D. Aitchison: firstname.lastname@example.org; or Richard
A. Rachubinski: email@example.com
Abbreviations used in this paper: GO, gene ontology; ORE, oleate-
The online version of this paper contains supplemental material.
Understanding complex cellular responses to external cues
demands a comprehensive analysis of the molecular networks
governing signaling, transcription, and morphology. Systems
biology provides an unprecedented opportunity to investigate
such complex dynamic networks and can enable a comprehen-
sive understanding of all the components governing a response
and the interplay among them. The peroxisome is an attractive
organelle to study using the approaches of systems biology.
Peroxisomes are rapidly affected in size, number, and protein
composition by changes in nutrients available to the cell, are
required for cell survival only under specifi c conditions, and are
relatively poorly understood with regard to their function and
biogenesis, although their dysfunction has been shown to cause
a variety of fatal or debilitating human conditions.
In yeast, peroxisomes are induced to proliferate in the
presence of fatty acids through a biogenic pathway initiated by
signaling events and transcriptional modulation preceding the
coordinated activities of ? 30 peroxins, which are collectively
responsible for organelle egression from the ER, matrix protein
import, and peroxisome division. Peroxisome biogenesis is a
sequential process, beginning with the insertion of the mem-
brane protein Pex3p into subdomains of the ER ( Hoepfner et al.,
2005 ) and ending with matrix protein entry into peroxisomes
( Erdmann and Schliebs, 2005 ; Leon et al., 2006 ) to obtain meta-
bolically active organelles. Yeast cells restrict the ? -oxidation of
fatty acids to peroxisomes ( Hiltunen et al., 2003 ), and func-
tional peroxisomes are therefore indispensable for yeast growth
on media containing fatty acids as the principal carbon source.
The mechanisms through which environmental cues are
transduced into activation of the peroxisome biogenic pathway
remain largely unknown. These mechanisms could regulate per-
oxisome biogenesis at the level of the transcriptional status of genes
encoding peroxisomal proteins, the traffi cking of proteins and
lipids that are destined to become constituents of peroxisomes,
tases and kinases required for peroxisome biogenesis
is the fi rst genome-wide analysis of phosphorylation
events controlling organelle biogenesis. We evaluate
signaling molecule deletion strains of the yeast Saccha-
romyces cerevisiae for presence of a green fl uorescent
protein chimera of peroxisomal thiolase, formation of
peroxisomes, and peroxisome functionality. We fi nd
that distinct signaling networks involving glucose-mediated
gene repression, derepression, oleate-mediated induction,
eversible phosphorylation is the most common
posttranslational modifi cation used in the regula-
tion of cellular processes. This study of phospha-
and peroxisome formation promote stages of the bio-
genesis pathway. Additionally, separate classes of sig-
naling proteins are responsible for the regulation of
peroxisome number and size. These signaling networks
specify the requirements of early and late events of
peroxisome biogenesis. Among the numerous signaling
proteins involved, Pho85p is exceptional, with func-
tional involvements in both gene expression and peroxi-
some formation. Our study represents the fi rst global
study of signaling networks regulating the biogenesis of
Genome-wide analysis of signaling networks
regulating fatty acid – induced gene expression
and organelle biogenesis
Ramsey A. Saleem , 1 Barbara Knoblach , 2 Fred D. Mast , 2 Jennifer J. Smith , 1 John Boyle , 1 C. Melissa Dobson , 2
Rose Long-O ’ Donnell , 1 Richard A. Rachubinski , 2 and John D. Aitchison 1,2
1 Institute for Systems Biology, Seattle, WA 98103
2 Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
JCB • VOLUME 181 • NUMBER 2 • 2008 282
of glucose but is also not induced by the removal of glucose.
The third state is an induced state in which an ORE is bound
by the transcription factors Oaf1p and Pip2p in response to fatty
acid stimulation. The range of activation of the POT1 promoter is
over 1,000-fold, making this promoter comparable to the most
strongly regulated promoters ( Navarro and Igual, 1994 ). In the
repressed state, the POT1 promoter is expressed at only ? 0.4%
of the level of its induced state. In the derepressed state, the ex-
pression level is 14% ( Einerhand et al., 1991 ). Similar condition-
specifi c differences in the levels of the POT1 gene product are
seen by Northern and Western analyses. A genome-wide analysis
of loss-of-function kinase and phosphatase mutant strains of
S. cerevisiae allowed us the opportunity to assess the contribu-
tions made by individual kinases and phosphatases to the induction
of oleate-responsive genes, such as POT1 , and to peroxisome bio-
genesis. To identify signaling networks that are responsible for
glucose derepression, oleate induction, and peroxisome biogenesis,
we measured the expression of a peroxisomal reporter chimera,
Pot1p-GFP, and the organellar biogenic response in signaling
gene-deletion mutants grown in glucose, glycerol, and oleate.
This study represents the fi rst comprehensive investigation of ki-
nase and phosphatase activity in the complex biological process
of organelle biogenesis.
A collection of 249 kinase and phosphatase gene deletion strains
of S. cerevisiae expressing a chromosomally integrated gene fu-
sion encoding Pot1p and GFP was evaluated for the expression
of the chimera and the formation and functionality of peroxisomes.
We addressed four parameters with respect to these processes:
glucose repression, glycerol-mediated derepression, oleate induc-
tion, and peroxisome morphology. By incubating each signaling
molecule deletion strain in glucose-, glycerol-, or oleate-containing
and/or the biological activities of proteins required for assembly,
maturation, division, and turnover of peroxisomes. The prolif-
erative capacity of peroxisomes coincides with the fatty acid –
responsive transcriptional regulation of many of the genes encoding
peroxisomal proteins ( Karpichev and Small, 1998 ; Smith et al.,
2002 ) and involves the transcriptional activators Adr1p, Oaf1p,
and Pip2p ( Rottensteiner et al., 1996 ; for review see Gurvitz
and Rottensteiner, 2006 ). Oaf1p and Pip2p bind directly to
oleate-responsive elements (OREs) in the promoter region of co -
ordinately responsive peroxisomal matrix proteins (for review
see Gurvitz and Rottensteiner, 2006 ). Adr1p is necessary for full
ORE-mediated activation, as it directly activates PIP2 and ORE-
containing targets ( Rottensteiner et al., 2003 ; Smith et al., 2007 ;
for review see Gurvitz and Rottensteiner, 2006 ). Although Oaf1p
appears to bind fatty acids directly, which in turn could directly
activate this factor ( Baumgartner et al., 1999 ; Phelps et al., 2006 ),
the intracellular signaling networks that lead to the composite
coordinated response resulting in the induction of peroxisomal
proteins and the formation of the peroxisome itself are unknown.
Global transcriptome studies demonstrate that many per-
oxisomal matrix proteins respond coordinately to oleic acid
( Koerkamp et al., 2002 ; Smith et al., 2002 , 2007 ; for review see
Gurvitz and Rottensteiner, 2006 ). We therefore used peroxisomal
3-ketoacyl-CoA thiolase (Pot1p), a peroxisomal matrix protein,
as a prototype of peroxisomal matrix enzymes to query the ef-
fects of systematic deletions of signaling proteins in the yeast
Saccharomyces cerevisiae . The POT1 promoter exists predomi-
nantly in three states depending on the available carbon source
( Einerhand et al., 1991 ; Igual et al., 1992 ). Glucose repression is
dominant to both derepression and induction ( Einerhand et al.,
1991 ; Igual et al., 1992 ). Addition of oleate in the presence of
glucose does not lead to the expression of genes encoding peroxi-
somal proteins. The second state is derepression. In the de-
repressed state, the promoter is no longer repressed by the presence
Figure 1. Glucose repression. (A) Pot1p-GFP
levels in strains that are deleted for signaling
molecules and that display an increase in
Pot1p-GFP levels in glucose, which is indica-
tive of a defect in glucose-mediated repression
of the POT1 locus. The y axis is shown on a
log 10 scale. (B) Plot of Pot1p-GFP fl uorescence
levels detected by FACS analysis of deletion
strains with high levels of Pot1p-GFP in glucose
but that exhibit a paucity of Pot1p-GFP after
incubation in oleate-containing medium for 6 h.
(C) A core of six signaling molecules whose
deletion results in a severe defect in the ability
of cells to express Pot1p-GFP in either glycerol
or oleate after 6 h of incubation. These strains
likely represent positive effectors of the transi-
tion from a glucose-repressed state to either a
derepressed or oleate-induced state. The heat
maps in A and C show the relative intensities
of Pot1p-GFP fl uorescence between mutants
and wild-type cells in the four media condi-
tions tested, with red and green indicating an
increase and a decrease in fl uorescence, re-
spectively. Error bars show SD.
283 CONTROL OF PEROXISOME DYNAMICS BY PHOSPHORYLATION • SALEEM ET AL.
derepressed or an oleate-induced state. The panel of deletion
strains was incubated in glucose-containing medium and then
either in glycerol- or oleate-containing medium for 6 h ( Fig. 1 C ).
Six genes ( SNF4 , YPL236c , PIF1 , SIT4 , LCB5 , and VPS34 )
encoding signaling molecules were identifi ed that, when deleted,
caused dramatic defects in the ability of cells to transition from
a glucose-repressed state regardless of whether this transition
occurred in glycerol or oleate ( > 1 and 2 SD, respectively, below
the population mean). That the same group of mutants was identi-
fi ed upon glycerol derepression or oleate induction suggests
that the same process is illuminated in each case. We therefore
attribute the phenotype of these mutants to their inability to ef-
fectively derepress the glucose state.
In contrast, deletions of three genes ( PSR2 , TEL1 , and
CDC5 ) were found to cause dramatic defects in oleate-induced
expression but were not found to signifi cantly (1 SD below the
population mean) affect glycerol derepression. We consider these
genes to be positive effectors of the oleate response ( Fig. 2 A ,
heat map). These results show that, in addition to well studied
glucose repression and derepression activities, there are specifi c
signaling events required for oleate-mediated induction. We thus
consider the core of the response to involve stepwise progres-
sion from glucose repression to glycerol depression to oleate
induction, culminating in peroxisome biogenesis.
In addition to the three effectors with the most dramatic
effects on oleate-mediated induction discussed in the previous
paragraph, we also identifi ed a larger group of oleate-specifi c
positive effectors whose absence caused less pronounced reduc-
tions in Pot1p-GFP levels (between 1 and 2 SD below that of
population mean; Fig. 2 B ) but nonetheless were not identifi ed as
signifi cant in the glycerol dataset (1 SD below the mean; Table S1).
medium and then measuring levels of Pot1p-GFP by FACS
analysis (Table S1, available at http://www.jcb.org/cgi/content/
full/jcb.200710009/DC1), we were able to assign each signal-
ing molecule as a positive, negative, or neutral effector of ex-
pression of Pot1p-GFP. By imaging analysis, we were also able
to determine the effect that each gene deletion had on peroxi-
some biogenesis after induction of peroxisomes in oleate.
The POT1 gene is normally repressed by glucose. Deletions
that result in increased levels of Pot1p-GFP in glucose represent
signaling proteins that function as positive effectors of glucose
repression. This group includes cax4 ? , ssn3 ? , elm1 ? , hrr25 ? ,
pho85 ? , dbf2 ? , ctk1 ? , and yck3 ? ( Fig. 1 A ). Northern blot
analysis in representative deletion strains demonstrates that the
Pot1p-GFP reporter refl ects mRNA levels expressed from POT1
and another typical ORE-containing gene (Fig. S1 B, available
This group of mutants defective in glucose repression can
be further divided into three subgroups ( Fig. 1 B ). The fi rst
group ( elm1 ? , yck3 ? , and cax4 ? ) has increased expression
in all conditions tested (glucose, glycerol, and oleate media).
The second group ( ssn3 ? and ctk1 ? ) we term “ uncoupled. ” These
strains do not repress well in glucose and also respond poorly
to oleate induction. The third group includes hrr25 ? , pho85 ? ,
and dbf2 ? strains. These cells respond poorly during the fi rst
6 h of oleate-mediated induction but, unlike the uncoupled
strains, Pot1p-GFP expression levels in these mutants increase
moderately in response to oleate treatment and eventually reach
wild-type levels (20 h; Fig. S2, available at http://www.jcb
We next sought to identify genes responsible for the tran-
sition from the glucose-repressed state to either a glycerol-
Figure 2. Oleate-specifi c positive effectors.
(A) Pot1p-GFP levels in deletion mutants that
result in a signifi cant oleate-specifi c decrease in
the levels of Pot1p-GFP ( > 2 SD). A strain deleted
for PIP2 is included as a control for negative
effectors. The heat map shows that decreases
in Pot1p-GFP levels are most pronounced after
6 h of oleate incubation. (B) Genes required
for effi cient oleate induction. Pot1p-GFP ex-
pression levels of deletion strains that showed
decreases in Pot1p-GFP levels of between
1 and 2 SD below the population mean are
shown. The heat map shows the relative inten-
sities of Pot1p-GFP fl uorescence among these
strains under the four media conditions tested,
with red and green indicating an increase and
a decrease in fl uorescence, respectively.
JCB • VOLUME 181 • NUMBER 2 • 2008 284
glucose repression. To do so, deletion strains were incubated in
a medium combining 2% glucose and 1% oleate. Remark-
ably, none of the deletions tested showed a signifi cantly in-
creased level of Pot1p-GFP beyond that detected in the presence
of glucose alone (unpublished data).
The phenotypes of the panel of deletion strains were also
examined to defi ne the role of individual members of the collec-
tion in peroxisome biogenesis. Peroxisome formation was ob-
served by confocal fl uorescence microscopy over a period of
20 h (Fig. S2). In wild-type cells, peroxisomes became visible as
punctate structures by 2 h of incubation in oleate and proliferated
extensively over time. To assess peroxisome formation, micro-
scopic images were used to determine peroxisome volume and
number, as well as peroxisome-specifi c fl uorescence intensity, as
distinct from overall cellular fl uorescence intensity determined
by FACS analysis ( Fig. 4 A ). Although the majority of mutants
that were unable to produce normal peroxisomes were also iden-
tifi ed by FACS analysis, morphological examination of the panel
identifi ed additional classes of signaling proteins required for
normal peroxisome biogenesis. None of the mutants showed an
observable mislocalization of the Pot1p-GFP reporter. Cluster
analysis of the peroxisomal features associated with a panel of
225 deletion mutants revealed the presence of four dominant
classes of mutant phenotypes ( Fig. 4 B ): Cluster I, with fewer and
enlarged peroxisomes; Cluster II, with increased number but
smaller volume of peroxisomes; Cluster III, with no detectable
peroxisomes; and Cluster IV, with few small peroxisomes.
Membership in Clusters III and IV corresponds closely to
mutants identifi ed by FACS analysis. Cluster III represents
those deletion strains that are unable to effi ciently form peroxi-
somes and constitute positive effectors of peroxisome biogenesis.
Interestingly, this group includes Snf1p. Snf1p has previously
been shown to be required by cells to overcome glucose-mediated
repression and to induce peroxisomes ( Igual et al., 1992 ; Simon
et al., 1992 ; Navarro and Igual, 1994 ) and the POT1 gene in par-
ticular ( Simon et al., 1992 ). Thus, although snf1 ? could be con-
sidered a “ gold standard, ” POT1 expression was not signifi cantly
different in snf1 ? cells compared with wild-type cells upon
transition to glycerol, and the quantitative analysis reported here
identifi ed mutants that had more dramatic defects in Pot1p-
GFP expression during derepression and oleate induction.
Collectively, these data suggest that there is a core group of genes
required for derepression and activation of oleate-induced genes
but that the coordination of effi cient cellular responses is contrib-
uted to differing extents by condition-specifi c effectors.
We also identifi ed a group of negative regulators whose de-
letion caused an increase in the expression of Pot1p-GFP in ole-
ate ( Fig. 3 A ). This group ( ark1 ? , hxk2 ? , kin3 ? , cdc19 ? , cla4 ? ,
hsl1 ? , and cln3 ? ) does not include those mutants that show in-
creased levels of Pot1p-GFP in glucose ( Fig. 1 A ) but rather
those deletion strains that show normal glucose-repressed levels
of Pot1p-GFP before the transition to an oleate-induced state
( Fig. 3 A ). In line with their roles as negative regulators, the popu-
lation variability of these deletion strains is high ( Fig. 3 A , right).
Likewise, a set of deletion strains exhibited increased Pot1p-GFP
fl uorescence ( > 1 SD above wild-type levels) upon transition to
glycerol ( Fig. 3 B ). It is noteworthy that only HSL1 negatively
regulates both processes, again suggesting common stepwise ele-
ments to the derepression and activation of POT1 in the context
of coordination with other cellular processes.
Finally, we tested the panel of strains to search for those
deletions that might allow cells to bypass the dominance of
Figure 3. Negative effectors. (A) Oleate neg-
ative effectors. Pot1p-GFP levels in deletion
mutants that exhibit an increase in Pot1p-GFP
after 3 h of incubation in oleate are shown.
At 6 h, the increase in Pot1p-GFP levels are
generally higher than the population mean but
show increased variability (right). (B) Glycerol
negative effectors. The increase in Pot1p-GFP
detected in deletion strains after 6 h of incuba-
tion in glycerol-containing medium is shown.
Only the hsl1 ? mutant shows an up-regulation
of Pot1p-GFP in both oleate and glycerol me-
dia, indicating that the normal role for HSL1 is
as a negative effector in both carbon sources.
The heat map shows the relative intensities of
Pot1p-GFP fl uorescence in these strains under
the four media conditions tested, with red and
green indicating an increase and a decrease in
fl uorescence, respectively. Error bars show SD.
285 CONTROL OF PEROXISOME DYNAMICS BY PHOSPHORYLATION • SALEEM ET AL.
and cax4 ? cells. Although these mutants cluster adjacent to
one another with respect to peroxisome morphology, they have
distinctly different phenotypes with respect to Pot1p-GFP ex-
pression. Cells deleted for REG1 are defective in producing
Pot1p-GFP, whereas cax4 ? cells show increased production of
Pot1p-GFP in glucose ( Fig. 1 A ).
Cluster I contains cells with fewer and enlarged peroxi-
somes. Of the 26 genes represented in Cluster I, 9 (35%) genes
were found to also function in regulation of the Pot1p-GFP lev-
els either positively or negatively ( ARK1 , CLN3 , DBF2 , HSL1 ,
PHO85 , TEL1 , TPS2 , YCK3 , and PHO80 ). Deletions of ARK1
and PHO85 also regulate the size of the peroxisomes, with both
deletions resulting in increases in peroxisomal volume with
concomitant decreases in peroxisomal numbers. Peroxisomes
in pho85 ? cells appear at early time points of oleate induction
and, after extended incubation, result in large peroxisomal
structures (20 h of oleate induction; Fig. 5 A ). Similar, but less
dramatic, results were observed in sip1 ? cells (Fig. S2). These data
suggest that Pho85p and Sip1p act as repressors of peroxisome
These correspond to strains identifi ed by FACS analysis to
have the largest decreases in Pot1p-GFP levels, with the excep-
tion of the pex3 ? strain, which cannot make peroxisomes and
was included as a control. Cluster IV represents those strains
that show peroxisome biogenesis phenotype defects resulting
in a few small peroxisomes. These are deletions with FACS
scores between 1 and 2 SD below wild-type levels but that over
a 20-h time course are still able to form peroxisomal structures.
Importantly, membership in this group also includes seven ad-
ditional genes that were not identifi ed as weak responders at
the early time points investigated by FACS. We attribute the
detection of these genes to the longer time periods used for
Clusters I and II reveal genes involved in biogenesis that
could not be distinguished by FACS analysis alone. Cluster II
represents those deletions that show increases in the numbers of
peroxisomes present with modest effects on peroxisomal volume.
The value and complementary nature of this analysis is illus-
trated by examining the Pot1p-GFP expression data for reg1 ?
Figure 4. Image analysis. (A) Scatter plot of Pot1p-GFP fl uorescence intensity of peroxisomes (arbitrary units) versus the number of peroxisomes per μ m 2 .
The mean peroxisomal volume ( μ m 3 ) of each strain is also indicated. Blue circles are used to highlight those mutant strains referred to in the text, whereas
gray circles are used to show the remaining population. (B) K-means clustering of standardized values for cell size, peroxisome volume, and number of
peroxisomes per cell, with associated microscopic images of selected deletion strain cells at 20 h of oleate incubation. Bar, 10 ? m. Clusters are discussed
in the text. Cluster V is presented in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200710009/DC1).
JCB • VOLUME 181 • NUMBER 2 • 2008 286
and two thirds of these strains also showed morphological de-
fects ( Fig. 5 D ). Of the 64 strains with morphological defects,
approximately half had fatty acid metabolism defects. Thus, it
is diffi cult to predict functionality based on morphology alone.
Indeed, although pho85 ? and yak1 ? cells have similar peroxi-
somal morphology defects, they have distinctly different myris-
tic acid metabolism properties. This is similar to the situation
with classically defi ned pex mutants. Although the fi rst pex mu-
tants were identifi ed by their inability to grow on fatty acids,
many of the pex mutants identifi ed more recently are able to
metabolize fatty acids and, thus, do not have major peroxisomal
assembly or protein import defects.
We have systematically and quantitatively surveyed the functional
consequences of deletions of the majority of the known yeast
kinases, phosphatases, and cyclins encoded by the S . cerevisiae
genome with respect to their roles as positive, negative, or neutral
biogenesis and that biogenesis and expression are coordinated
by the activity of pathways involving these proteins.
The remaining 17 deletion strains of Cluster 1 also had
fewer enlarged peroxisomes but were not detected to aberrantly
affect POT1 expression. This includes yak1 ? cells, which ap-
peared to have few lobate peroxisomes after 8 h of oleate incu-
bation. This interesting phenotype was confi rmed and expanded
upon by ultrastructural analyses ( Fig. 5 B ). In contrast to the
wild-type strain and another Cluster 1 strain, pho85 ? , peroxi-
somes of yak1 ? cells were observed to cluster and to sometimes
share a common membrane ( Fig. 5 B ).
Peroxisome functionality was assessed by growth of cells
on solid medium (YPBM) containing the fatty acid myristate
as the principal carbon source ( Smith et al., 2006 ). Cells with
a functional peroxisomal ? -oxidation system grow on YPBM,
and their consumption of myristate can be assessed by the forma-
tion of haloes around colonies ( Fig. 5 C ; and Table S2, available
49 mutants were unable to effi ciently metabolize myristic acid,
Figure 5. Peroxisome biogenesis. (A) Time course of peroxisome biogenesis over 20 h of incubation in oleate-containing YPBO medium. At 8 h, yak1 ?
cells exhibit elongated peroxisomes, whereas pho85 ? cells present detectable peroxisomes before the oleate induction process (t = 0), and peroxisomes
develop as smaller structures as compared with peroxisomes in wild-type cells. Bar, 10 ? m. (B) Electron micrographs of peroxisomes in the wild-type strain
BY4742 and the pho85 ? and yak1 ? deletion strains. Cells deleted for PHO85 show peroxisomes of reduced size, whereas yak1 ? cells show clustering of
peroxisomes and thickened membranes between adherent peroxisomes. Bar, 0.25 μ m. (C) Growth of strains on myristate-containing YPBM medium reveals
reduced fatty acid metabolism in pex3 ? , snf1 ? , and pho85 ? strains. (D) Venn diagram showing the relationships between the number of deletion strains
with myristate utilization defects, peroxisome morphology defects, and both defects.
287 CONTROL OF PEROXISOME DYNAMICS BY PHOSPHORYLATION • SALEEM ET AL.
an oleate-activated state. This study identifi es signaling machin-
eries that specify the requirements of the early and late biogenesis
events of peroxisomes and groups of global regulators of glucose
repression, cell cycle pathways that differentially activate genes
encoding peroxisomal proteins, and mediators controlling the
phosphorylation status of lipids. These results are summarized in
Fig. 6 , which presents systems level schematics of signaling pro-
tein networks controlling oleate-mediated induction of Pot1p-
GFP and peroxisome biogenesis ( Table I ).
Glucose-mediated repression of the Pot1p-GFP peroxisomal
reporter requires eight of the signaling molecules tested ( Fig. 6 A ).
Five were specifi cally required for repression ( Fig. 6 A , cir-
cled), and three (Ctk1p, Pho85p, and Ssn3p) were also impli-
cated in oleate activation. Snf1p is a well characterized protein
required to overcome glucose repression ( Igual et al., 1992 ;
Navarro and Igual, 1994 ). Accordingly, the positive effectors
effectors of oleate-mediated peroxisome biogenesis during oleate
activation. To evaluate the specifi c role of each signaling protein,
the process has been dissected into four fundamental steps: re-
pression of genes for peroxisome-related proteins by glucose,
derepression by transfer of cells to glycerol, oleate-mediated acti-
vation of gene expression, and production of the organelle itself.
Our fi ndings indicate that although the majority of signaling pro-
teins tested do not signifi cantly affect the process, specifi c and
common key signaling proteins regulate each of the four steps.
To place these data in the context of our current understanding of
the roles of each protein, networks of signifi cant signaling mole-
cules in these processes were overlaid with the physical and
genetic interactions between each signaling component ( Fig. 6 )
together with the Gene Ontology (GO) annotations (https://www
.proteome.com/proteome/YPD) for these proteins, which were used
as an aid for understanding their reported roles. Oleate-induced
peroxisomal biogenesis is preceded by signaling mechanisms to
transition cells in a stepwise fashion from a glucose-repressed to
Figure 6. Core signaling molecules of Pot1p
expression and peroxisome biogenesis. (A) Rep-
resentation of the genes discussed in the
text showing the oleate activation network.
Included are genes involved in glucose repres-
sion, together with the overlap between the
glycerol derepression and oleate activation
datasets. Genes are represented by shapes,
whereas interactions between gene products
are represented by lines. For both A and B,
lines are shown as black for physical inter-
actions and blue for genetic interactions. In-
creasing thickness of lines refl ects increasing
published interactions. Genes required for
repression in glucose are shown by squares,
whereas reporter levels in the deletion strain
of a given gene in glycerol are indicated by
the oval borders. Reporter levels of the dele-
tion strains after incubation in oleate are rep-
resented by circles. A color gradient between
green and red is used to represent the strength
effect of the gene products on expression of
Pot1p-GFP for both oleate activation and glu-
cose repression. Red is used for those gene
products that function to reduce the levels of
Pot1p-GFP, whereas green is used for those
gene products whose normal function is to
increase the levels of Pot1p-GFP. A color gra-
dient between blue and yellow is used to rep-
resent the strength of the effect of these gene
products on Pot1p-GFP after 6 h of glycerol
incubation. Blue is used to indicate those gene
products that function as positive effectors of
Pot1p-GFP expression, whereas yellow is used
to indicate those gene products that function
as negative effectors of Pot1p-GFP expression.
(B) Representation of the genes that form the
peroxisome biogenesis network. Genes are
represented by circles and interactions be-
tween gene products are represented by lines.
Peroxisome volume is indicated by circles,
whereas peroxisome number is indicated by
the circle border. A color gradient between
green and red circles represents the strength of
regulation of peroxisome volume, whereas a
color gradient between blue and yellow circle
borders represents the strength of regulation of peroxisome number. Those gene products which are negative effectors of peroxisome volume are shown in
red, whereas those that are positive effectors are shown in green. Those gene products which are negative effectors of peroxisome number are represented
by yellow circle borders, whereas those that are positive effectors are represented by blue circle borders. PHO85 is included in this network because of
its role in biogenesis.
JCB • VOLUME 181 • NUMBER 2 • 2008 288
peroxisomes themselves (see Biogenesis). Ssn3p and Ctk1p
are components of the RNA polymerase II complex and, in
agreement with the data presented here, have previously been
shown to both positively and negatively infl uence gene ex-
pression ( Liao et al., 1995 ; Hengartner et al., 1998 ).
Most genes encoding peroxisomal proteins are repressed in glu-
cose and activated in oleic acid ( Smith et al., 2002 ). We con-
sider the transition between these states to involve two steps,
i.e., depression followed by activation. To identify proteins in-
volved in each of these steps, cells were incubated in glucose
and then glycerol to identify genes involved in derepression and
in glucose and then oleic acid to identify genes involved in both
derepression and activation. Proteins involved in both processes
of repression consist of several Snf1p-interacting proteins, in-
cluding Ssn3p, Ctk1p, Elm1p, Pho85p, and Hrr25p ( Fig. 6 A ),
as well as two proteins, Dbf2p and Yck3p, which are separated
with respect to their interaction with Snf1p by intermediary pro-
teins. The role of Cax4p in glucose repression is less clear, as it has
not been previously implicated in this process and is function-
ally annotated as being involved in sphingolipid metabolism.
Deletions of most genes in this class of positive effectors
of glucose repression are unaffected by oleate induction. This
is not surprising, because the glucose-repressed and oleate-
induced states are separable. However, unlike other members
of the group, strains defi cient in Pho85p, Ssn3p, and Ctk1p are
defective in both glucose repression and oleate-mediated in-
duction. Pho85p plays complex roles in repression and induc-
tion of the POT1 locus, as well as a role in biogenesis of
Table I. Gene deletions producing a phenotype in a given process (repression, depression, induction, or biogenesis) grouped according to their
roles at a systems level
Positive process effectors cax4
Negative process effectors ctk3
289 CONTROL OF PEROXISOME DYNAMICS BY PHOSPHORYLATION • SALEEM ET AL.
factors, Oaf1p, Pip2p, Adr1p, and Oaf3p, governing the response.
In this network, Adr1p induces expression of PIP2 . Pip2p forms
a heterodimer with constitutively present Oaf1p, which is acti-
vated by binding fatty acids directly ( Phelps et al., 2006 ). Oaf3p
appears to modulate the response ( Smith et al., 2007 ). The sig-
naling response to oleate is less well characterized. It is apparent
from the data shown here that in addition to Oaf1p binding oleate,
a host of signaling proteins is required for effi cient induction
of fatty acid – responsive genes and peroxisome formation itself.
For example, Psr2p is a plasma membrane phosphatase that, along
with Whi2p, is thought to modulate the general stress response
by regulating the activity of Msn2p ( Kaida et al., 2002 ). The ac-
tivity of Psr2p does not appear to be important for depression
but is required for oleate-mediated induction. The general stress
response is tightly integrated with the oleate response ( Koerkamp
et al., 2002 ; Smith et al., 2002 ), and our data suggest that Psr2p
is an important molecular link between these two processes.
Several other highly connected proteins are also required
for oleate induction, but not glycerol derepression, of the Pot1p-
GFP reporter ( Fig. 6 A ). As expected, this group includes compo-
nents that interact with Snf1p, but it also includes a surprising
number of proteins involved in regulation of the cell cycle (Cdc5p,
Cln3p, Gin4p, Hsl1p, Kin3p, Mck1p, Pho80p, Pho85p, Sap185p,
Ste20p, and Vhs3p). This may refl ect a dramatic shift in the meta-
bolic state of cells in glycerol as compared with oleate, as well as
the coordination required for an extended cell cycle ( ? 1.5 – 2 h in
glucose vs. > 6 h in oleate; Koerkamp et al., 2002 ; Smith et al.,
2002 ). The other major annotation in this network is that of phos-
phatidylinositol metabolism, which contains four genes required
for oleate induction. These proteins function as phosphatidylino-
sitol kinases in a variety of processes, including actin polym-
erization (Inp52p), telomere maintenance (Tel1p), transcriptional
control (Tor1p), and actin polarity and membrane targeting
(Cla4p). The precise role of these proteins in the context of oleate
induction is unclear; however, it is clear that they function to en-
hance oleate responsiveness. From our data, we conclude that co-
ordinated activation of the glucose regulation, cell cycle regulation,
and phosphatidylinositol metabolism networks is necessary for
effi cient expression of oleate-induced peroxisomal proteins.
Microscopy was used to identify those signaling proteins re-
quired for the formation of morphologically normal peroxisomes.
Because peroxisome formation lies downstream of gene tran-
scription, in our analysis, we subtracted the signaling proteins
involved in the transcriptional response from the entire reper-
toire of signaling proteins to identify those proteins that spe-
cifi cally effect peroxisome formation ( Fig. 6 B and Table I ).
We refer to this group as biogenesis-specifi c effectors. Three
primary subgroups (i.e., those involved in actin regulation or
phosphatidylinositol metabolism and “ other, ” those proteins
with disparate GO annotations of effectors) were revealed by
this analysis. There are six actin regulating molecules (Inp51p,
Mkk1p, Mkk2p, Pph21p, Rom1p, and Sac1p), and much of the
function of these signaling proteins is mediated through the ac-
tion of Rho regulators. Rho1p has been shown to localize to,
and be required for, the normal biogenesis of peroxisomes
are therefore considered responsible for transition to the de-
repressed state, whereas proteins involved in only oleate induc-
tion are considered specifi c to induction.
Glycerol-mediated derepression, unlike glucose repression,
is mediated by signaling proteins whose GO annotations are dis-
tributed between numerous processes, for example, vacuolar pro-
tein sorting, cell cycle, phosphatidylinositol signaling, and
sphingolipid biosynthesis. The lack of specifi city in the annota-
tions may speak to the numerous processes that are preceded by
relief of glucose repression, complexity of the transition to the
derepressed state, the lack of publicly available information with
regards to this process, or all three points. As discussed in the pre-
vious section, Snf1p is well characterized as being required for
expression of glucose-repressed genes upon derepression, includ-
ing the POT1 gene ( Einerhand et al., 1991 ; Igual et al., 1992 ;
Simon et al., 1992 ). Although we detect considerable defects in
this transition for snf1 ? mutants, this analysis identifi ed six pro-
teins whose absence results in defects more severe than those ob-
served for snf1 ? cells. Snf4p is the ? -subunit of the active Snf1p
kinase complex ( Simon et al., 1992 ; Elbing et al., 2006 ). Vps34p
is involved in glucose repression of the ADH2 gene encoding
alcohol dehydrogenase ( Voronkova et al., 2006 ), whereas Sit4p,
Lcb5p, Pif1p, and Ypl236p have not previously been implicated
in the expression of glucose-repressed genes.
The derepression component of the oleate activation net-
work ( Fig. 6 A ) reveals previously uncharacterized roles for
signaling molecules, including the sphingolipid metabolism
kinase Lcb5p and a phosphatidylinositol kinase involved in
vacuolar transport, Vps34p. Sphingolipids are a class of lipids that
are known to play roles in signal transmission and are impor-
tant regulators of the cell cycle (for reviews, see Hannun and
Obeid, 2002 ; Fernandis and Wenk, 2007 ). Phosphatidylinositols
are, like sphingolipids, becoming recognized as more than struc-
tural components of lipid bilayers. They can be metabolized
quickly and the products act as second messengers (for review,
see Strahl and Thorner, 2007 ). Our data show that Lcb5p and
Vps34p are critical effectors for the transition of the POT1 lo-
cus to a derepressed or induced state. Furthermore, these data
show that genes annotated in processes as diverse as sphingo-
lipid metabolism, phosphatidylinositol metabolism, and vacu-
olar protein sorting play signifi cant roles in derepression.
YPL236c is a previously uncharacterized gene whose de-
letion has a profound effect on Pot1p-GFP expression during
derepression. High-throughput screens have localized the protein
to mitochondria and the vacuole ( Kumar et al., 2002 ; Huh et al.,
2003 ). Ypl236p might interact with Ufd2p, an E4 ligase involved
in multiubiquitination and can be phosphorylated and palmi-
toylated ( Zhu et al., 2000 ; Roth et al., 2006 ). No function has been
attributed to Ypl236p, but based on the data presented here, we
propose that Ypl236p is an important positive effector of transcrip-
tion of glucose-repressed genes upon derepression or induction.
We and others have begun to characterize the transcriptional
regulatory network of oleate induction in S. cerevisiae ( Smith et al.,
2002 , 2007 ; for review see Gurvitz and Rottensteiner, 2006 ).
These studies have focused on the function of four transcription
JCB • VOLUME 181 • NUMBER 2 • 2008 290
An exception to this rule is Pho85p, which plays a complex
role in repression, induction, and biogenesis. In glucose condi-
tions, levels of Pot1p-GFP are high in pho85 ? cells, which is
in good agreement with confocal data (Table S2), demonstrat-
ing a role for Pho85p as a positive effector of glucose-mediated
repression. By 6 h of oleate incubation, the levels of Pot1p-
GFP in pho85 ? cells are still 1 SD below those observed in
wild-type cells, yet visual analysis revealed the formation of
multiple small peroxisomes (Fig. S2 and Table S2), implying
that peroxisome biogenesis is premature in the pho85 ? strain
and that Pho85p acts as a negative effector of peroxisome bio-
genesis. After extended periods of oleate induction, Pot1p-GFP
levels in pho85 ? cells approach those of wild-type cells, and
by 20 h in YPBO, the multiple small peroxisomes found in
pho85 ? cells cluster and present as large peroxisomes by fl uo-
rescence microscopy ( Fig. 4 A ) and as multiple adjacent small
peroxisomes by EM ( Fig. 5 C ). Thus, Pho85p appears to act as
a positive effector of glucose-mediated repression of Pot1p-
GFP expression (hence the up-regulation of Pot1p-GFP in
pho85 ? cells in glucose medium), a positive effector of effi -
cient Pot1p-GFP expression during oleate induction (hence the
diminished levels of Pot1p-GFP in pho85 ? cells after 6 h in
oleate medium), and a negative effector of biogenesis (hence
the premature appearance of peroxisomes in pho85 ? cells in
oleate medium). These data show that Pho85p is a unique pro-
tein in the overall process, and its various functions remain fer-
tile ground for further investigation.
An interesting feature revealed by our data is the possibil-
ity of a feedback mechanism between peroxisomes and the nu-
cleus. In pex3 ? cells in which no peroxisomal structures can be
observed, there is a signifi cant decrease in the levels of Pot1p-
GFP after 6 h of oleate induction ( Fig. 2 B ). This observation
raises the intriguing possibility that effi cient transcription, at
least initially, may be regulated by the peroxisome itself; how-
ever, further study is required.
Despite recent advances in the analysis of the kinomes of
various organisms ( Pelkmans et al., 2005 ; Ptacek et al., 2005 ),
this study is, to our knowledge, the fi rst global investigation of
both kinase and phosphatase activities in a complex biological
phenomenon, the biogenesis of an organelle. This work is also the
fi rst study of the signaling mechanisms underlying the develop-
ment of an organelle from gene transcription through to the for-
mation of intact mature and functioning structures. By using a
systematic and quantitative approach, we have delineated the sig-
naling proteins required for glucose-mediated transcriptional re-
pression, transition from a repressed to a derepressed state in
glycerol and oleate, induction in response to oleate exposure, and
biogenesis of peroxisomes with respect to the size, number, and
timing of the development of the organelle. The dynamics of
peroxisomes in wild-type cells and the complexity of phenotypes
uncovered in our strain collection are suggestive of a stringent
integration of signaling events in the temporal and spatial control
of peroxisome assembly. Collectively, our data demonstrate both
the complexity and convergence of signaling pathways in organ-
elle biogenesis. This work thus provides a framework from which
the stepwise function of these pathways and processes can be in-
vestigated and determined.
through its regulation of the polymerization state of actin on
the peroxisome ( Marelli et al., 2004 ). With respect to the second
major subgroup involved in phosphatidylinositol metabolism
(Ckl1p, Cmk1p, Inp51p, Kss1p, Sac1p, Sps1p, and Vps34p), it
is interesting to note that certain phosphoinositides have been
shown to be required for peroxisomal precursor fusion ( Boukh-
Viner et al., 2005 ). This fact, and the diminutive size of peroxi-
somes in strains such as kss1 ? , sac1 ? , and vps34 ? , whose
encoded proteins have lipid kinase and phosphatase activities
(in addition to the actin regulatory activity of both Sac1p and
Inp51p), suggest that these peroxisomal forms may be trapped
in an immature stage in early biogenesis. As peroxisomal fu-
sion is a poorly characterized process, the mutant phenotypes
detected here provide candidate molecules for investigation to-
ward a mechanistic understanding of the process. The large
number of biogenesis effector proteins with disparate GO
annotations likely refl ects the complexity of biogenesis and the
host of processes required for development of the organelle.
The phenotypes identifi ed by confocal microscopy are
similar to those observed in peroxisome division mutants, i.e.,
a few large peroxisomes or many small peroxisomes ( Fig. 4 B ,
Clusters I and II) or are mutants with no or little detectable induc-
tion of peroxisomes ( Fig. 4 B , Clusters III and IV), which possibly
represents mutants with very low levels of Pot1p-GFP. These data
suggest that, in particular for proteins in clusters I and II, biogen-
esis that is regulated by this group of signaling proteins involves
growth and division rather than de novo biogenesis of peroxi-
somes from the ER ( Hoepfner et al., 2005 ; Tam et al., 2005 ).
However, defects at the early stages of biogenesis, for example,
fi ssion from the ER, cannot be ruled out at this point, and feed-
back between the ER and proteins involved in peroxisome bio-
genesis remains an uninvestigated possibility.
None of the signaling mutants tested led to cytosolic ac-
cumulation of Pot1p-GFP, suggesting that peroxisomal matrix
protein import, per se, is not altered. However, there are at least
two pathways for matrix protein import. PTS2-containing pro-
teins like Pot1p are imported by Pex7p, whereas PTS1-containing
proteins are imported by Pex5p and membrane proteins are
integrated into the organelle by yet additional mechanisms
(for reviews see Rayapuram and Subramani, 2006 ; Platta and
Erdmann, 2007 ). Therefore, it is possible that some of the mutants
examined have PTS1, membrane protein, or cargo-specifi c per-
oxisomal protein targeting, transport, or membrane protein inte-
gra tion defects.
The size and abundance of peroxisomes can be regulated
by the levels of the matrix proteins they contain ( Chang et al.,
1999 ; Smith et al., 2000 , van Roermund et al., 2000 ). In our
data, we see evidence of this phenomenon. For example, the in-
creased levels of Pot1p-GFP seen in the cax4 ? strain result in a
dramatic increase in the number of peroxisomes, whereas cells
deleted for SNF1 or SNF4 , among other genes, show decreased
levels of Pot1p-GFP together with concomitant decreases in
peroxisome volume and number ( Fig. 5 B and Fig. S2). As men-
tioned previously, because these mutants are affected in their
Pot1p-GFP levels, they were not included as biogenic effectors,
a group that we restrict to mutants that specifi cally exhibit al-
tered peroxisome morphology.
291 CONTROL OF PEROXISOME DYNAMICS BY PHOSPHORYLATION • SALEEM ET AL.
clustering, choosing 10 clusters as the initial parameter. Similar clusters
were merged, leaving a total of fi ve clusters.
Interaction data were obtained through the Saccharomyces Genome Data-
base (http://www.yeastgenome.org). Interaction data for each group
( Table I ) were acquired using the Saccharomyces Genome Database batch
download tool on December 8, 2007. High-throughput physical inter-
actions were removed from the interaction dataset. Interactions were visu-
alized using Cytoscape 2.5.1 ( Shannon et al., 2003 ).
Online supplemental material
Fig. S1 shows that the regulation of peroxisomal loci is conserved. Fig. S2
shows confocal images of oleate induction of the Pot1p-GFP chimera in the
deletion strains over a 20-h time course. Fig. S3 shows the k-means cluster-
ing of peroxisome number and volume determined by analysis of confocal
images. Table S1 contains the data for the FACS analysis of the deletion
strains. Table S2 contains the data for the imaging-based quantifi cation
of peroxisome biogenesis. Table S3 lists the S. cerevisiae strains used in
this study. Online supplemental material is available at http://www.jcb
We thank Drs. Andrew Simmonds and Xuejun Sun for help with image analy-
sis. The technical assistance of Honey Chan, Hanna Kroliczak, Richard Poirier,
Elena Savidov, Dwayne Weber, and Mark Fuller and the computational assis-
tance of Dorian Rachubinski are greatly appreciated.
This work was supported by grant 53326 from the Canadian Institutes
of Health Research to R.A. Rachubinski and by grants RO1 GM067228,
RR022220, and PM50 GMO76547 from the National Institutes of Health to
J.D. Aitchison. R.A. Saleem is a recipient of a Canadian Institutes for Health
Research Postdoctoral Fellowship. C.M. Dobson is the recipient of a Full-Time
Fellowship from the Alberta Heritage Fund for Medical Research. R.A. Rachu-
binski is Canada Research Chair in Cell Biology and an International Research
Scholar of the Howard Hughes Medical Institute.
Submitted: 1 October 2007
Accepted: 24 March 2008
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Materials and methods
Yeast cell culture and genetic manipulation
All S. cerevisiae strains used in this study are listed in Table S3 (available
at http://www.jcb.org/cgi/content/full/jcb.200710009/DC1). Strains
were cultured at 30 ° C in the following media: YPD (1% yeast extract, 2%
peptone, and 2% glucose), YPBO (0.3% yeast extract, 0.5% peptone,
0.5% potassium phosphate buffer, pH 6.0, 0.5% Tween 40, and 1% oleic
acid), YPBM (0.67% yeast nitrogen base, 0.1% yeast extract, 0.5% potas-
sium phosphate buffer, pH 6.0, 0.5% Tween 40, and 0.125% myristic
acid), YPBD (0.67% yeast nitrogen base, 0.1% yeast extract, 0.5% potas-
sium phosphate buffer, pH 6.0, 0.5% Tween 40, and 2% glucose), and
SSM (Complete supplement medium [BIO101], 0.09% KH 2 PO 4 , 0.023%
K 2 HPO 4 , 0.05% MgSO 4 , and 0.35% (NH 4 ) 2 SO 4 ). To construct strains ex-
pressing the Pot1p-GFP chimera, the POT1 gene was tagged at its 3 ? end
through homologous recombination with a PCR-based strategy in frame
with the sequence encoding Aequorea victoria GFP ( Scholz et al., 2000 ).
All genomic integrations were confi rmed by PCR. Integrations were also
verifi ed by sequencing. Allelism for strains with signifi cant phenotypes, as
measured here, was established by complementation analysis.
Cells of deletion strains were grown in YPBD to mid-log phase in 96-well
deep-well plates, pelleted, washed with water, resuspended in the same
volume of YPBO or YPBG, and incubated with shaking for 3 or 6 h as indi-
cated at 30 ° C. Cells were then processed by removal of the medium, fi xa-
tion in 3.7% formaldehyde for 30 min, and resuspension in water and
analyzed with a FACSCalibur (BD Biosciences) with the following parame-
ters: forward scatter, E0 haploid linear scale; side scatter, 520 V linear
scale; fl uorescence, 490 V logarithmic scale. Cells were loaded onto the
FACSCalibur using the high throughput sampler (BD Biosciences). The high
throughput sampler was run in standard mode using a 96-well fl at-bottomed
plate and was set to sample 10 μ l at a rate of 2 μ l/s. All experiments were
replicated a minimum of three times with a minimum of two technical repli-
cates per biological replicate.
Confocal fl uorescence microscopy
To visualize the Pot1p-GFP chimera in time-course experiments, strains
were grown to midexponential phase in YPBD, resuspended in the same
volume of YPBO, and incubated for 2, 4, 6, 8, and 20 h in YPBO at 30 ° C.
Five images per time point per strain were captured randomly with a Plan-
Apochromat 63 × /1.4 NA oil differential interference contrast objective on
an inverted microscope (Axiovert 200; Carl Zeiss, Inc.) equipped with a
confocal scanner (LSM 510 META; Carl Zeiss, Inc.). GFP was excited with
a 488-nm laser and its emission was collected using a 505-nm long-pass
fi lter. Images were captured at 23 ° C with the microscope pinhole adjusted
to 1 Airy unit. Total peroxisome induction and peroxisome volumes were
determined with Metamorph Imaging System 6.3 software (MDS Analyti-
cal Technologies). The channels of the LSM images were split, the transmis-
sion channel was processed with “ Flatten Background ” and “ Median ”
fi lters and binarized, and the total and mean cell areas in the image plane
were recorded for each strain. Peroxisome induction was calculated as
pixel intensity of the green channel per cell. Peroxisome volumes were re-
corded in the green channel using Metamorph ’ s “ threshold image ” and
“ count cells ” functions. Peroxisome numbers per cell at 20 h of induction
were manually counted in at least 100 cells per strain using the spot count-
ing tool of the Imaris 5.0 imaging software (Bitplane). All other computa-
tional analyses were performed with Origin 7.0 software (OriginLab).
Fixation and processing of cells for EM were performed as described previ-
ously ( Eitzen et al., 1997 ).
Pot1p-GFP fl uorescence. Data were converted by standardizing the raw log
fl uorescence for each experiment according to the equation Z = (X - ? )/ ? ,
where X = mean log fl uorescence of a given deletion, ? = the population
mean fl uorescence, and ? = the population mean SD. Heat maps were
generated using the mean standardized values in the TMEV program
( Saeed et al., 2003 , 2006 ) and standardized using a range of ? 3 and 3
for all FACS data.
Imaging data. Imaging data were comprised of cell area, peroxi-
some volume, and number of peroxisomes for each deletion strain after
20 h of induction in YPBO. Values were standardized using the equation
in the previous section and clustered with the TMEV program using k-means
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