A collection of 249 kinase and phosphatase gene deletion strains of S. cerevisiae
expressing a chromosomally integrated gene fusion 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 induction, and peroxisome morphology. By incubating each signaling molecule deletion strain in glucose-, glycerol-, or oleate-containing 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 signaling molecule as a positive, negative, or neutral effector of expression of Pot1p-GFP. By imaging analysis, we were also able to determine the effect that each gene deletion had on peroxisome biogenesis after induction of peroxisomes in oleate.
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 cax4Δ
, and yck3Δ
(). Northern blot analysis in representative deletion strains demonstrates that the Pot1p-GFP reporter reflects mRNA levels expressed from POT1
and another typical ORE-containing gene (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200710009/DC1
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 indicative of a defect in glucose-mediated repression of the POT1 locus. The y axis is shown (more ...)
This group of mutants defective in glucose repression can be further divided into three subgroups (). The first group (elm1Δ
, and cax4Δ
) has increased expression in all conditions tested (glucose, glycerol, and oleate media). The second group (ssn3Δ
) we term “uncoupled.” These strains do not repress well in glucose and also respond poorly to oleate induction. The third group includes hrr25Δ
, and dbf2Δ
strains. These cells respond poorly during the first 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.org/cgi/content/full/jcb.200710009/DC1
We next sought to identify genes responsible for the transition from the glucose-repressed state to either a glycerol-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 (). Six genes (SNF4, YPL236c, PIF1, SIT4, LCB5, and VPS34) encoding signaling molecules were identified 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 identified 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 effectively 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 significantly (1 SD below the population mean) affect glycerol derepression. We consider these genes to be positive effectors of the oleate response (, heat map). These results show that, in addition to well studied glucose repression and derepression activities, there are specific signaling events required for oleate-mediated induction. We thus consider the core of the response to involve stepwise progression from glucose repression to glycerol depression to oleate induction, culminating in peroxisome biogenesis.
Figure 2. Oleate-specific positive effectors. (A) Pot1p-GFP levels in deletion mutants that result in a significant oleate-specific decrease in the levels of Pot1p-GFP (>2 SD). A strain deleted for PIP2 is included as a control for negative effectors. The (more ...)
In addition to the three effectors with the most dramatic effects on oleate-mediated induction discussed in the previous paragraph, we also identified a larger group of oleate-specific positive effectors whose absence caused less pronounced reductions in Pot1p-GFP levels (between 1 and 2 SD below that of population mean; ) but nonetheless were not identified as significant in the glycerol dataset (1 SD below the mean; Table S1). 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 particular (Simon et al., 1992
). Thus, although snf1Δ
could be considered a “gold standard,” POT1
expression was not significantly different in snf1Δ
cells compared with wild-type cells upon transition to glycerol, and the quantitative analysis reported here identified 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 efficient cellular responses is contributed to differing extents by condition-specific effectors.
We also identified a group of negative regulators whose deletion caused an increase in the expression of Pot1p-GFP in oleate (). This group (ark1Δ, hxk2Δ, kin3Δ, cdc19Δ, cla4Δ, hsl1Δ, and cln3Δ) does not include those mutants that show increased levels of Pot1p-GFP in glucose () but rather those deletion strains that show normal glucose-repressed levels of Pot1p-GFP before the transition to an oleate-induced state (). In line with their roles as negative regulators, the population variability of these deletion strains is high (, right). Likewise, a set of deletion strains exhibited increased Pot1p-GFP fluorescence (>1 SD above wild-type levels) upon transition to glycerol (). It is noteworthy that only HSL1 negatively regulates both processes, again suggesting common stepwise elements to the derepression and activation of POT1 in the context of coordination with other cellular processes.
Figure 3. Negative effectors. (A) Oleate negative 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 (more ...)
Finally, we tested the panel of strains to search for those deletions that might allow cells to bypass the dominance of glucose repression. To do so, deletion strains were incubated in a medium combining 2% glucose and 1% oleate. Remarkably, none of the deletions tested showed a significantly increased 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 define the role of individual members of the collection in peroxisome biogenesis. Peroxisome formation was observed by confocal fluorescence 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, microscopic images were used to determine peroxisome volume and number, as well as peroxisome-specific fluorescence intensity, as distinct from overall cellular fluorescence intensity determined by FACS analysis (). Although the majority of mutants that were unable to produce normal peroxisomes were also identified by FACS analysis, morphological examination of the panel identified 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 (): 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.
Figure 4. Image analysis. (A) Scatter plot of Pot1p-GFP fluorescence intensity of peroxisomes (arbitrary units) versus the number of peroxisomes per μm2. The mean peroxisomal volume (μm3) of each strain is also indicated. Blue circles are used to (more ...)
Membership in Clusters III and IV corresponds closely to mutants identified by FACS analysis. Cluster III represents those deletion strains that are unable to efficiently form peroxisomes and constitute positive effectors of peroxisome biogenesis. These correspond to strains identified by FACS analysis to have the largest decreases in Pot1p-GFP levels, with the exception of the pex3Δ 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 additional genes that were not identified 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 microscopical analysis.
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 illustrated by examining the Pot1p-GFP expression data for reg1Δ and cax4Δ cells. Although these mutants cluster adjacent to one another with respect to peroxisome morphology, they have distinctly different phenotypes with respect to Pot1p-GFP expression. Cells deleted for REG1 are defective in producing Pot1p-GFP, whereas cax4Δ cells show increased production of Pot1p-GFP in glucose ().
Cluster I contains cells with fewer and enlarged peroxisomes. Of the 26 genes represented in Cluster I, 9 (35%) genes were found to also function in regulation of the Pot1p-GFP levels 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 pho85Δ cells appear at early time points of oleate induction and, after extended incubation, result in large peroxisomal structures (20 h of oleate induction; ). Similar, but less dramatic, results were observed in sip1Δ cells (Fig. S2). These data suggest that Pho85p and Sip1p act as repressors of peroxisome biogenesis and that biogenesis and expression are coordinated by the activity of pathways involving these proteins.
Figure 5. Peroxisome biogenesis. (A) Time course of peroxisome biogenesis over 20 h of incubation in oleate-containing YPBO medium. At 8 h, yak1Δ cells exhibit elongated peroxisomes, whereas pho85Δ cells present detectable peroxisomes before the (more ...)
The remaining 17 deletion strains of Cluster 1 also had fewer enlarged peroxisomes but were not detected to aberrantly affect POT1 expression. This includes yak1Δ cells, which appeared to have few lobate peroxisomes after 8 h of oleate incubation. This interesting phenotype was confirmed and expanded upon by ultrastructural analyses (). In contrast to the wild-type strain and another Cluster 1 strain, pho85Δ, peroxisomes of yak1Δ cells were observed to cluster and to sometimes share a common membrane ().
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 β-oxidation system grow on YPBM, and their consumption of myristate can be assessed by the formation of haloes around colonies (; and Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200710009/DC1
). 49 mutants were unable to efficiently metabolize myristic acid, and two thirds of these strains also showed morphological defects (). Of the 64 strains with morphological defects, approximately half had fatty acid metabolism defects. Thus, it is difficult to predict functionality based on morphology alone. Indeed, although pho85Δ
cells have similar peroxisomal morphology defects, they have distinctly different myristic acid metabolism properties. This is similar to the situation with classically defined pex
mutants. Although the first pex
mutants were identified by their inability to grow on fatty acids, many of the pex
mutants identified more recently are able to metabolize fatty acids and, thus, do not have major peroxisomal assembly or protein import defects.