Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

Interaction of Pik1p and Sjl proteins in membrane trafficking

Phosphatidylinositol (PtdIns) phosphates are involved in signal transduction, cytoskeletal organization, and membrane traffic. PtdIns 4-phosphate [PtdIns(4)P], produced in yeast by PtdIns 4-kinase (Pik1p), appears to regulate Golgi secretory function. PtdIns(4)P is also produced by dephosphorylation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], catalyzed by one of the three yeast Sjl proteins, homologs of the mammalian synaptic vesicle-associated PtdIns(4,5)P2 5-phosphatase, synaptojanin. To determine whether Pik1p and Sjl proteins operate in the same pathway or regulate the same process, we used a genetic approach. Mutation in the PIK1 gene displays synthetic genetic interactions with deletions of individual SJL genes. Deletion of SJL3 gene is synthetically lethal with pik1ts, and deletions of SJL1 or SJL2 genes in pik1ts cells exacerbate the temperature sensitivity, neomycin sensitivity, and defect in invertase secretion. A diminished level of PtdIns(4)P and increased level of PtdIns(4,5)P2 in pik1(ts)sjl1delta and pik1(ts)sjl2delta cells, compared with pik1ts cells, indicate that PtdIns(4)P is specifically required for secretion. Collectively, our results suggest that Pik1p and the Sjl proteins coordinately function to regulate the dynamic phosphorylation-dephosphorylation of the polar heads of phosphoinositides, and this process appears to be important for membrane trafficking pathways.     

Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases, STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 or PIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly, pik1(ts) cells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4(ts) cells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by approximately 50%; however, stt4(ts)/pik1(ts) double mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P(2). The aberrant Golgi morphology found in pik1(ts) mutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4(ts) cells at restrictive temperature result in a dramatic change in vacuole size compared with pik1(ts) cells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P(2) that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.     

Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-β) was necessary for nuclear entry, but not Kap60 (importin-α), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH₂ terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Δ cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Δ10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Δ cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Δ cells. However, coexpression of Pik1(Δ10-192) and Pik1-CCAAX in pik1Δ cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.     

Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1(ts) allele and one of two temperature-sensitive PtdIns 4-kinase genes, stt4(ts) or pik1(ts), we have demonstrated that the bulk of PtdIns(4)P that accumulates in sac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated with stt4(ts) but not pik1(ts) mutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1(ts) mutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.     

Essential and distinct roles of phosphatidylinositol 4-kinases,Pik1p and Stt4p,in yeast autophagy

Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome–vacuole fusion in yeast cells, respectively.     

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.     

Homotypic secretory vesicle fusion induced by the protein tyrosine phosphatase MEG2 depends on polyphosphoinositides in T cells

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P(3) binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P(3) synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P(3) in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.     

Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.     

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.     

Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.     

Evidence for an intrinsic toxicity of phosphatidylcholine to Sec14p-dependent protein transport from the yeast Golgi complex

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient "bypass Sec14p" phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1(ts)-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.     

Nte1p-mediated deacylation of phosphatidylcholine functionally interacts with Sec14p

Deciphering the function of the essential yeast Sec14p protein has revealed a regulatory interface between cargo secretion from Golgi and lipid homeostasis. Abrogation of the CDP-choline (CDP-Cho) pathway for phosphatidylcholine (PC) synthesis allows for life in the absence of the otherwise essential Sec14p. Nte1p, the product of open reading frame YML059c, is an integral membrane phospholipase against CDP-Cho-derived PC producing intracellular glycerophosphocholine (GPCho) and free fatty acids. We monitored Nte1p activity through in vivo PC turnover measurements and observed that intracellular GPCho accumulation is decreased in a sec14(ts) strain shifted to 37 degrees C in 10 mm choline (Cho)-containing medium compared with a Sec14p-proficient strain. Overexpression of two Sec14p homologs Sfh2p and Sfh4p in sec14(ts) cells restored secretion and growth at the restrictive temperature but did not restore GPCho accumulation. Instead, newly synthesized PC was degraded by phospholipase D (Spo14p). Similar analysis performed in a sec14Delta background confirmed these observations. These results imply that the ability of Sfh2p and Sfh4p to restore secretion and growth is not through a shared function with Sec14p in the regulation of PC turnover via Nte1p. Furthermore, our analyses revealed a profound alteration of PC metabolism triggered by the absence of Sec14p: Nte1p unresponsiveness, Spo14p activation, and deregulation of Pct1p. Sfh2p- and Sfh4p-overexpressing cells coped with the absence of Sec14p by controlling the rate of phosphocholine formation, limiting the amount of Cho available for this reaction, and actively excreting Cho from the cell. Increased Sfh4p also significantly reduced the uptake of exogenous Cho. Beyond the new PC metabolic control features we ascribe to Sfh2p and Sfh4p we also describe a second role for Sec14p in mediating PC homeostasis. Sec14p acts as a positive regulator of Nte1p-mediated PC deacylation with the functional consequence of increased Nte1p activity increasing the permissive temperature for the growth of sec14(ts) cells.     

Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Deltasac1 Deltainp54 and sac1ts Deltainp54 mutants. Furthermore, sac1ts Deltainp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Deltasac1 Deltainp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.     

Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components

BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting. RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p. CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.     

The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles

Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.     

Phosphatidylinositol 4-Kinases Are Required for Autophagic Membrane Trafficking

Macroautophagy (hereafter autophagy) is a degradative cellular pathway that protects eukaryotic cells from stress, starvation, and microbial infection. This process must be tightly controlled because too little or too much autophagy can be deleterious to cellular physiology. The phosphatidylinositol (PtdIns) 3-kinase Vps34 is a lipid kinase that regulates autophagy, but the role of other PtdIns kinases has not been examined. Here we demonstrate a role for PtdIns 4-kinases and PtdIns4P 5-kinases in selective and nonselective types of autophagy in yeast. The PtdIns 4-kinase Pik1 is involved in Atg9 trafficking through the Golgi and is involved in both nonselective and selective types of autophagy, whereas the PtdIns4P 5-kinase Mss4 is specifically involved in mitophagy but not nonselective autophagy. Our data indicate that phosphoinositide kinases have multiple roles in the regulation of autophagic pathways.     

A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast

SEC4, one of the 10 genes involved in the final stage of the yeast secretory pathway, encodes a ras-like, GTP-binding protein. In wild-type cells, Sec4 protein is located on the cytoplasmic face of both the plasma membrane and the secretory vesicles in transit to the cell surface. In all post-Golgi blocked sec mutants, Sec4p is predominantly associated with the secretory vesicles that accumulate as a result of the secretory block. Sec4p is synthesized as a soluble protein that rapidly (t1/2 less than or equal to 1 min) and tightly associates with secretory vesicles and the plasma membrane by virtue of a conformational change of a covalent modification. These data suggest that Sec4p may function as a "G" protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane.     

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.