Inositol Starvation Research Articles

Establishment and inheritance of epigenetic transcriptional memory.

For certain inducible genes, the rate and molecular mechanism of transcriptional activation depends on the prior experiences of the cell. This phenomenon, called epigenetic transcriptional memory, accelerates reactivation and requires both changes in chromatin structure and recruitment of poised RNA Polymerase II (RNAPII) to the promoter. Forms of epigenetic transcriptional memory have been identified in S. cerevisiae, D. melanogaster, C. elegans, and mammals. A well-characterized model of memory is found in budding yeast where memory of inositol starvation involves a positive feedback loop between gene-and condition-specific transcription factors, which mediate an interaction with the nuclear pore complex and a characteristic histone modification: histone H3 lysine 4 dimethylation (H3K4me2). This histone modification permits recruitment of a memory-specific pre-initiation complex, poising RNAPII at the promoter. During memory, H3K4me2 is essential for recruitment of RNAPII and faster reactivation, but RNAPII is not required for H3K4me2. Unlike the RNAPII-dependent H3K4me2 associated with active transcription, RNAPII-independent H3K4me2 requires Nup100, SET3C, the Leo1 subunit of the Paf1 complex and can be inherited through multiple cell cycles upon disrupting the interaction with the Nuclear Pore Complex. The H3K4 methyltransferase (COMPASS) physically interacts with the potential reader (SET3C), suggesting a molecular mechanism for the spreading and re-incorporation of H3K4me2 following DNA replication. Thus, epigenetic transcriptional memory is a conserved adaptation that utilizes a heritable chromatin state, allowing cells and organisms to alter their gene expression programs in response to recent experiences over intermediate time scales.

Mitotically heritable, RNA polymerase II-independent H3K4 dimethylation stimulates INO1 transcriptional memory.

For some inducible genes, the rate and molecular mechanism of transcriptional activation depend on the prior experiences of the cell. This phenomenon, called epigenetic transcriptional memory, accelerates reactivation, and requires both changes in chromatin structure and recruitment of poised RNA polymerase II (RNAPII) to the promoter. Memory of inositol starvation in budding yeast involves a positive feedback loop between transcription factor-dependent interaction with the nuclear pore complex and histone H3 lysine 4 dimethylation (H3K4me2). While H3K4me2 is essential for recruitment of RNAPII and faster reactivation, RNAPII is not required for H3K4me2. Unlike RNAPII-dependent H3K4me2 associated with transcription, RNAPII-independent H3K4me2 requires Nup100, SET3C, the Leo1 subunit of the Paf1 complex and, upon degradation of an essential transcription factor, is inherited through multiple cell cycles. The writer of this mark (COMPASS) physically interacts with the potential reader (SET3C), suggesting a molecular mechanism for the spreading and re-incorporation of H3K4me2 following DNA replication.

Alpha-arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis.

Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

Inositol Depletion Induced by Acute Treatment of the Bipolar Disorder Drug Valproate Increases Levels of Phytosphingosine

Bipolar disorder (BD) is a severe psychiatric illness affecting ∼1% of the world population. Valproate (VPA) and lithium, widely used for the treatment of BD, are not universally effective. These drugs have been shown to cause inositol depletion, but translating this observation to a specific therapeutic mechanism has been difficult, hampering the development of more effective therapies. We have shown previously in yeast that chronic VPA treatment induces the unfolded protein response due to increasing ceramide levels. To gain insight into the mechanisms activated during acute VPA treatment, we performed a genome-wide expression study in yeast treated with VPA for 30 min. We observed increased mRNA and protein levels of RSB1, which encodes an exporter of long chain bases dihydrosphingosine (DHS) and phytosphingosine (PHS), and further saw that VPA increased sensitivity of an rsb1Δ mutant to PHS, suggesting that VPA increases long chain base levels. Consistent with this, PHS levels were elevated in wild type and, to a greater extent, in rsb1Δ cells. Expression of ORM genes (negative regulators of PHS synthesis) and of fatty acid elongase genes FEN1 and SUR4 were decreased, and expression of YOR1 (exporter of PHS-1P) and DPL1 (lyase that degrades DHS-1P and PHS-1P) was increased. These effects were more pronounced in medium lacking inositol, and were mirrored by inositol starvation of an ino1Δ mutant. These findings provide a metabolic explanation as to how VPA-mediated inositol depletion causes increased synthesis of PHS and further support the therapeutic relevance of inositol depletion as a bipolar disorder treatment.

Calnexin is essential for survival under nitrogen starvation and stationary phase in Schizosaccharomyces pombe.

Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further shedding light on the multiple roles of calnexin.

Activation of Protein Kinase C-Mitogen-activated Protein Kinase Signaling in Response to Inositol Starvation Triggers Sir2p-dependent Telomeric Silencing in Yeast

Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.

Localization of Opi1pFFAT to lipid droplets during inositol starvation in Saccharomyces cerevisiae

Phosphatidic acid (PA) is a signaling lipid that plays an important role in mediating phospholipid biosynthesis. However, the subcellular distribution of PA remains elusive. In this study, PA levels were manipulated by shifting cells from inositol‐containing medium to inositol‐lacking medium. A mutated form of the Opi1p repressor, Opi1pFFAT, which loses the interaction with the endoplasmic reticulum (ER) protein Scs2p but contains a PA‐binding domain, relocalizes to lipid droplets (LDs) after 2‐h of inositol starvation. Opi1pFFAT localization was also monitored in mutant cells with defects in triacylglycerol (TAG) metabolism, including pah1Δ and lro1Δdga1Δ for TAG biosynthesis, and tgl3Δtgl4Δtgl5Δ for TAG mobilization. Opi1pFFAT localized to LDs in the tgl3Δtgl4Δtgl5Δ mutant, but did not localize to LDs in cells unable to synthesize TAG. Another mutated form of Opi1p, Opi1pFFAT/PAc4m, which carries mutations in both FFAT and PA‐binding domains, was also examined. Opi1pFFAT/PAc4m did not localize to LDs, but remained in the nucleus. These results suggest Opi1pFFAT binds to a specific pool of PA that is actively involved in TAG synthesis. Fld1p, a human seipin homolog in yeast that binds to the junction of LDs and ER, was shown to colocalize with Opi1pFFAT followed by 2‐h inositol starvation, suggesting a pool of PA to the sites of LD biogenesis. Supported by NIH grant GM019629.

Involvement of Seipin in Directing Localized PA for Lipid Droplet Formation

A loss of function of seipin, an endoplasmic reticulum (ER) membrane protein causes a severe congenital generalized lipodystrophy in humans. In yeast, deletion of seipin, FLD1, results in aberrant lipid droplets. However, the molecular mechanism of seipin is still unknown. Fei et al. (2011) reported that the deletion of FLD1 results in an increase in membrane phosphatidic acid (PA) concentration. In this study, we use Opi1p, a transcription repressor involved in phospholipid synthesis, as a PA sensor. Opi1p is known to bind to PA as well as the ER resident protein Scs2p. We report that Opi1p‐GFP forms puncta in the absence of FLD1. These puncta, usually next to droplets, are more pronounced with an Opi1p mutant (Opi1pFFAT) that cannot bind to Scs2p but still binds to PA. In order to test whether lipid droplets are necessary for the Opi1p‐GFP puncta in fld1Δ cells, we expressed Opi1p‐mCherry both in a mutant that cannot generate neutral lipids nor droplets (4KO; are1are2lro1dga1 KO), and one which also lacks FLD1 (5KO). Puncta were attenuated in these strains compared to fld1Δcells. In combination with the finding that Opi1p transiently localizes to droplets during inositol starvation (Chang et al, poster in this meeting), our data suggest that PA flux is important for droplet formation and that Fld1p facilitates utilization of PA during lipid droplet biogenesis. Supported by NIH grant GM084210.

Coordination of stress response signaling and phospholipid metabolism in yeast

Previously we showed that the availability of the lipid precursor inositol influences the levels and rates of synthesis and turnover of inositol phospholipids, phosphoinositides and complex sphingolipids. To discover the inositol‐dependent mechanisms that lead to activation of lipid‐mediated signaling pathways, we compared the simultaneous changes in lipid metabolism with those occurring in gene expression in yeast cells during a 3‐hour period of inositol starvation followed by a 30‐min period of inositol add‐back. We found that inositol starvation and add‐back led to dramatic changes in the abundance of inositol‐containing phospholipids, including phosphatidylinositol, the phosphoinositides, and complex sphingolipids. These changes were accompanied by the altered expression of hundreds of genes involved in diverse stress response pathways. Significantly we found that many mutants deleted for nonessential genes in the same stress response pathways exhibit inositol auxotrophy. These results suggest that activation of stress response signaling pathways is coordinated with inositol‐containing phospholipid metabolism.

Desumoylation of the endoplasmic reticulum membrane VAP family protein Scs2 by Ulp1 and SUMO regulation of the inositol synthesis pathway.

Posttranslational protein modification by the ubiquitin-like SUMO protein is critical to eukaryotic cell regulation, but much remains unknown regarding its operation and substrates. Here we report that specific mutations in the Saccharomyces cerevisiae Ulp1 SUMO protease, including its coiled-coil (CC) domain, lead to the accumulation of distinct sumoylated proteins in vivo. A prominent ~50-kDa sumoylated protein accumulates in a Ulp1 CC mutant. The protein was identified as Scs2, an endoplasmic reticulum (ER) membrane protein that regulates phosphatidylinositol synthesis and lipid trafficking. Mutation of lysine 180 of Scs2 abolishes its sumoylation. Notably, impairment of either cellular sumoylation or cellular desumoylation mechanisms inhibits cell growth in the absence of inositol and exacerbates the inositol auxotrophy caused by deletion of SCS2. Mutants lacking the Ulp2 SUMO protease are the most severely affected, and this defect was traced to the mutants' impaired ability to induce transcription of INO1, which encodes the rate-limiting enzyme of inositol biosynthesis. Conversely, inositol starvation induces a striking change in the profiles of total cellular SUMO conjugates. These results provide the first evidence of cross-regulation between the SUMO and inositol pathways, including the sumoylation of an ER membrane protein central to phospholipid synthesis and phosphoinositide signaling.

Endoplasmic reticulum involvement in yeast cell death

Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

Yet1p–Yet3p interacts with Scs2p–Opi1p to regulate ER localization of the Opi1p repressor

Lipid sensing mechanisms at the endoplasmic reticulum (ER) coordinate an array of biosynthetic pathways. A major phospholipid regulatory circuit in yeast is controlled by Scs2p, an ER membrane protein that binds the transcriptional repressor protein Opi1p. Cells grown in the absence of inositol sequester Scs2p-Opi1p at the ER and derepress target genes including INO1. We recently reported that Yet1p and Yet3p, the yeast homologues of BAP29 and BAP31, are required for normal growth in the absence of inositol. Here we show that the Yet1p-Yet3p complex acts in derepression of INO1 through physical association with Scs2p-Opi1p. Yet complex binding to Scs2p-Opi1p was enhanced by inositol starvation, although the interaction between Scs2p and Opi1p was not influenced by YET1 or YET3 deletion. Interestingly, live-cell imaging analysis indicated that Opi1p does not efficiently relocalize to the ER during inositol starvation in yet3Δ cells. Together our data demonstrate that a physical association between the Yet complex and Scs2p-Opi1p is required for proper localization of the Opi1p repressor to ER membranes and subsequent INO1 derepression.

Interruption of Inositol Sphingolipid Synthesis Triggers Stt4p-dependent Protein Kinase C Signaling

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.

Role of the transcription factor Opi1p and phosphatidic acid in the formation of lipid droplets in Saccharomyces cerevisiae

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644–1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. After the cells grew under inositol starvation for 2 hrs, novel punctate structures were recognized by Opi1p‐GFP in the FFAT and scs2 null mutants. Interestingly, these structures are highly associated with ER. Furthermore, by using Tgl4p‐RFP as a lipid droplet marker to identify these structures, we observed colocalization of Tgl4p‐RFP with the punctate structures. These results suggest a role for both Opi1p and PA in the formation of lipid droplets.Supported by NIH grant GM019629.

Calnexin Regulates Apoptosis Induced by Inositol Starvation in Fission Yeast

Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of calnexin has regulatory roles in apoptotic processes involving calnexin.

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

Function of Phosphatidylinositol in Mycobacteria

Phosphatidylinositol (PI) is an abundant phospholipid in the cytoplasmic membrane of mycobacteria and the precursor for more complex glycolipids, such as the PI mannosides (PIMs) and lipoarabinomannan (LAM). To investigate whether the large steady-state pools of PI and apolar PIMs are required for mycobacterial growth, we have generated a Mycobacterium smegmatis inositol auxotroph by disruption of the ino1 gene. The ino1 mutant displayed wild-type growth rates and steady-state levels of PI, PIM, and LAM when grown in the presence of 1 mM inositol. The non-dividing ino1 mutant was highly resistant to inositol starvation, reflecting the slow turnover of inositol lipids in this stage. In contrast, dilution of growing or stationary-phase ino1 mutant in inositol-free medium resulted in the rapid depletion of PI and apolar PIMs. Whereas depletion of these lipids was not associated with loss of viability, subsequent depletion of polar PIMs coincided with loss of major cell wall components and cell viability. Metabolic labeling experiments confirmed that the large pools of PI and apolar PIMs were used to sustain polar PIM and LAM biosynthesis during inositol limitation. They also showed that under non-limiting conditions, PI is catabolized via lyso-PI. These data suggest that large pools of PI and apolar PIMs are not essential for membrane integrity but are required to sustain polar PIM biosynthesis, which is essential for mycobacterial growth.

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.

Regulation of Profilin Localization in Saccharomyces cerevisiae by Phosphoinositide Metabolism

Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.