Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities
1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês
10.1093/emboj/17.17.4930
ISSN1460-2075
Autores Tópico(s)Erythrocyte Function and Pathophysiology
ResumoArticle1 September 1998free access Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities Andrew E. Wurmser Andrew E. Wurmser Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Scott D. Emr Corresponding Author Scott D. Emr Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Andrew E. Wurmser Andrew E. Wurmser Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Scott D. Emr Corresponding Author Scott D. Emr Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Author Information Andrew E. Wurmser1 and Scott D. Emr 1 1Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4930-4942https://doi.org/10.1093/emboj/17.17.4930 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40–50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases. Introduction The yeast vacuole is a hydrolytically active organelle that is functionally analogous to the mammalian lysosome (Klionsky et al., 1990). Proteins designated Vps (vacuolar protein sorting) mediate the transport of newly synthesized hydrolase precursors from the late Golgi to the vacuole via an intermediate endosome compartment (Vida et al., 1993). Mutations within VPS genes result in hydrolase mis-sorting and secretion as well as abnormal vacuole morphologies. Based on these mutant phenotypes, >40 VPS genes have been subgrouped into six major classes, A–F (Raymond et al., 1992). VPS34 is a class D gene which encodes a phosphatidylinositol 3-kinase (PtdIns 3-kinase) (Herman and Emr, 1990; Schu et al., 1993). Class D vps mutants exhibit severe defects in hydrolase sorting, contain enlarged vacuoles and cannot grow, or grow poorly, at elevated temperatures (Herman and Emr, 1990; Raymond et al., 1992). Vps34p phosphorylates phosphatidylinositol (PtdIns) at the D-3 position of the inositol ring and represents the only detectable PtdIns 3-kinase activity in Saccharomyces cerevisiae (Schu et al., 1993). Vps34p is recruited from the cytosol to the membrane-associated serine/threonine protein kinase, Vps15p (Herman et al., 1991; Stack et al., 1993). Although it is not yet clear how Vps15p is activated, Vps15p protein kinase activity is a prerequisite to the formation of a Vps15p–Vps34p complex (Stack et al., 1995). This interaction not only localizes Vps34p to the correct membrane compartment, but also stimulates its PtdIns 3-kinase activity >10-fold (Stack et al., 1995). Consistent with the role of Vps15p as an upstream regulator of Vps34p, inactivation of Vps15p results in severe decreases in cellular levels of phosphatidylinositol 3-phosphate [PtdIns(3)P] and also results in the mis-sorting of vacuole hydrolases (Herman et al., 1991; Stack et al., 1995). Vps34p is a member of a growing kinase family that phosphorylates PtdIns. These phosphorylated derivatives of PtdIns serve as second messengers which bind effector proteins, recruiting these proteins to specific subcellular localizations and/or influencing their activity (Toker and Cantley, 1997; Vanhaesebroeck et al., 1997). For example, 3-phosphorylated derivatives of PtdIns bind p85/p110 phosphoinositide 3-kinase, PDK1, Akt and the AP-2 adaptor complex, accounting, at least in part, for the effects of PI3-kinases on cell growth, apoptosis and membrane trafficking, respectively (Rameh et al., 1995; Franke et al., 1997; Rapoport et al., 1997; Stephens et al., 1998). Based on these and other studies, lipid kinases are recognized as an important part of the machinery that controls a diverse array of cellular functions via the formation of various phosphoinositide second messengers. PtdIns(3)P produced by Vps34p is also likely to function as a second messenger, although the relevant target(s) of PtdIns(3)P in yeast has not yet been identified. The discovery of specific cytoplasmic lipases and phosphatases which antagonize the activity of lipid kinases reveals the requirement of the cell to downregulate and modify signals mediated by phosphoinositides. For example, cleavage of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to distinct second messengers is carried out by phospholipase C in response to tyrosine-kinase- and G-protein-coupled receptor activation (Rhee, 1991; Sternweis and Smrcka, 1992). In addition, type II 5-phosphatases like synaptojanin and OCRL may also play roles in the turnover of PtdIns(4,5)P2 (Attree et al., 1992; McPherson et al., 1996). However, to date, 3-phosphorylated phosphoinositides have not been found to be good substrates of phospholipase C (Serunian et al., 1989; Woscholski et al., 1995). Instead, a family of proteins, including SHIPs, which exhibit phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity in vitro have been isolated (Woscholski and Parker, 1997). In addition, cytoplasmic 3-phosphatase activities have been detected (Woscholski et al., 1995) and purified (Caldwell et al., 1991). However, their precise role in the turnover of PtdIns(3)P is still unclear. In S.cerevisiae, it is likely that a specific turnover mechanism exists for PtdIns(3)P because PtdIns(3)P levels decrease significantly within 10 min after inactivation of Vps34p (Stack et al., 1995). Here, we present evidence demonstrating that PtdIns(3)P is degraded by an unexpected mechanism. We have found that interruption of endosome-to-vacuole transport leads to elevations in PtdIns(3)P levels by disrupting the turnover of PtdIns(3)P. Turnover of PtdIns(3)P is also dependent upon vacuolar hydrolase activity, strongly suggesting that PtdIns(3)P degradation occurs within the hydrolytic environment of the vacuole/lysosome lumen. We propose that a significant pool of PtdIns(3)P shifts from a cytoplasmic membrane leaflet where PtdIns(3)P is produced, to the lumen of the vacuole where it can be exposed to and turned over by active vacuolar enzymes. We present evidence indicating that lipids in the cytoplasmic leaflet of the membrane are transferred to the lumen of the vacuole through the invagination and budding of vesicles into the lumen of the vacuole or prevacuolar endosomes which then fuse with the vacuole. Inactivation of Vps34p results in the impaired fusion of endosomal transport intermediates with the vacuole. Therefore, PtdIns(3)P is not only a cargo of the endosome, but may also regulate this late step in the vacuolar protein transport pathway. Results Inactivation of the vacuolar t-SNARE, Vam3p, or the rab GTPase, Ypt7p, results in elevated PtdIns(3)P levels Vam3p (vacuolar t-SNARE) and Ypt7p (yeast rab7 GTPase) are essential factors in endosome-to-vacuole trafficking (Wichmann et al., 1992; Darsow et al., 1997). We have found that the mutation of VAM3 or YPT7 has strong effects on steady-state PtdIns(3)P levels in vivo. To assay PtdIns(3)P levels, SEY6210 (wild-type), TDY2 (vam3Δ) and WSY99 (ypt7Δ) cells were labeled with myo-[2-3H]inositol for 12 h, lysed, and the cellular lipids extracted with chloroform/methanol. Lipids were then deacylated, separated by HPLC (see Materials and methods), and the amount of radioactivity in each fraction collected was quantified. This system clearly separates PtdIns(3)P from phosphatidylinositol 4-phosphate [PtdIns(4)P] (Schu et al., 1993). Wild-type cells had ∼2200 c.p.m. of 3H incorporated into both PtdIns(3)P (fraction 33) and PtdIns(4)P (fraction 36), yielding a PtdIns(3)P: PtdIns(4)P ratio of 1.1 (Figure 1A). Deletion of VAM3 or YPT7 resulted in significant increases in PtdIns(3)P levels, without affecting PtdIns(4)P levels, which translate to 4.6- and 4.8-fold increases, in the PtdIns(3)P: PtdIns(4)P ratio, respectively. Figure 1.Cellular PtdIns(3)P levels increase in vam3 and ypt7 mutant strains. (A) Wild-type cells, vam3Δ cells and ypt7Δ cells were labeled with myo-[2-3H]inositol for 12 h, lysed and extracted with chloroform/methanol. After deacylation, lipids were separated by HPLC yielding peaks corresponding to PtdIns(3)P and PtdIns(4)P. (B) Wild-type cells and two aliquots of vam3tsf cells were labeled at 26°C with myo-[2-3H]inositol for 12 h. One aliquot of vam3tsf cells was maintained at the permissive temperature of 26°C while the second aliquot was shifted to the nonpermissive temperature of 38°C for the final 20 min of labeling. Wild-type cells were also shifted to 38°C for the final 20 min of labeling. Following cell lysis, lipids were extracted in chloroform/methanol, deacylated and analyzed by HPLC. Deacylated PtdIns(3)P and PtdIns(4)P peaks are indicated. The data presented are representative of multiple experiments. Download figure Download PowerPoint Previous work has described the isolation of a temperature-sensitive allele of the VAM3 gene which, at the nonpermissive temperature, inhibits the transport of hydrolases to the vacuole (Darsow et al., 1997). As an additional means of compromising Vam3p function, without causing secondary effects which may result from the deletion of VAM3, PtdIns(3)P levels were also measured in TDY2 + pVAM3-6.416 (vam3tsf) cells briefly shifted to the nonpermissive temperature. At the permissive temperature (26°C), vam3tsf cells exhibited wild-type PtdIns(3)P levels (Figure 1B). However, vam3tsf cells grown at 26°C for 12 h and then shifted to the nonpermissive temperature (38°C) for 20 min prior to the assay, exhibited a 2-fold increase in PtdIns(3)P and no change in PtdIns(4)P levels. Wild-type cells shifted to 38°C for 20 min did not exhibit a significant elevation in PtdIns(3)P. VAM3 and YPT7 are considered a subset of class B VPS genes (Darsow et al., 1997). We also tested whether inactivation of VPS33, a class C VPS gene that functions at a late stage of vacuolar protein sorting (Banta et al., 1990), had an effect on PtdIns(3)P levels. Similar to the vam3tsf, the LBY317 + pVPS33-8.416 (vps33tsf) strain when shifted to the nonpermissive temperature for 15 min resulted in a 2-fold increase in PtdIns(3)P levels (data not shown). These data indicate that the mutation of either VAM3, YPT7 or VPS33 results in increased PtdIns(3)P levels, perhaps by disrupting trafficking to the vacuole. Deletion of VAM3 and YPT7 do not influence Vps34p PtdIns 3-kinase activity Vps34p is the only detectable PtdIns 3-kinase in S.cerevisiae (Schu et al., 1993). This suggests that one possibility by which mutations of VAM3 and YPT7 may increase steady-state PtdIns(3)P levels is by increasing Vps34p activity. Alternatively, deletion of these genes may impair the turnover of PtdIns(3)P. To differentiate between these possibilities, protein extracts from wild-type, vam3Δ and ypt7Δ cells were assayed for relative levels of PtdIns 3-kinase activity (see Materials and methods). When incubated with PtdIns and [γ-32P]ATP, 2 μg of protein extracts from wild-type, vam3Δ and ypt7Δ strains all contained equal levels of PtdIns 3-kinase activity, suggesting that mutations in VAM3 and YPT7 do not increase steady-state PtdIns(3)P levels by stimulating Vps34p (Figure 2). In contrast, 2 μg of protein extract from cells co-overexpressing the VPS15 and VPS34 genes exhibited >10-fold increases in PtdIns 3-kinase activity under identical assay conditions and with the same film exposure (Figure 2). This suggests that deletion of VAM3 and YPT7 inhibits the turnover of PtdIns(3)P rather than increasing its synthesis. Figure 2.Vps34p PtdIns 3-kinase activity is not affected in vam3Δ or ypt7Δ mutant strains. Protein extracts were prepared from wild-type cells and strains deleted for VAM3 or YPT7. PtdIns 3-kinase activity present was measured by incubating 2 μg of protein extract with [γ-32P]ATP and sonicated PtdIns for 5 min at 25°C. The lipid products of this reaction were resolved by TLC and analyzed by autoradiography (equal film exposures are shown). PtdIns(3)P and PtdIns(4)P are indicated. Download figure Download PowerPoint Vacuolar hydrolase activity is required for PtdIns(3)P turnover Since endosomal transport intermediates fail to fuse with the vacuole and accumulate in the cytoplasm of vam3 and ypt7 mutants (Schimmoller and Riezman, 1993; Darsow et al., 1997), our data indicate that endosome-to-vacuole trafficking may be required for PtdIns(3)P turnover. One interpretation of these findings is that PtdIns(3)P could be degraded at the vacuole. This implies that deletion of either VAM3 or YPT7 may elevate PtdIns(3)P levels by spatially separating PtdIns(3)P in a prevacuolar compartment (e.g. endosome) from its turnover enzymes. If this hypothesis is correct, then the hydrolytic activity of the vacuole might be required for the turnover of PtdIns(3)P. PEP4, PRB1 and PRC1 encode the vacuolar proteinase A (PrA), proteinase B (PrB) and carboxypeptidase Y (CPY) hydrolases, respectively. PrA and PrB are important to the overall hydrolytic activity of the vacuole as they function to cleave proteolytically a variety of inactive vacuolar zymogens, including proteases (like CPY) and phosphatases (like alkaline phosphatase), to their mature, active forms (Klionsky and Emr, 1989; Hirsch et al., 1992; van den Hazel et al., 1992). Vacuolar enzymes can be separated into two broad classes: (i) PrA and PrB which activate other vacuolar enzymes; and (ii) the remaining proteases, lipases and phosphatases which, upon activation by PrA and/or PrB, carry out the bulk metabolism of their relevant substrates (Klionsky et al., 1990; van den Hazel et al., 1996). In order to test the possibility that PtdIns(3)P is turned over in a vacuolar hydrolase-dependent manner, steady-state PtdIns(3)P levels were measured in cells that are deficient in vacuolar hydrolase activity. Representative data from multiple analyses are shown in Figure 3. While wild-type cells had normal levels of PtdIns(3)P, TVY614 cells (pep4Δ/prb1Δ/prc1Δ), a strain simultaneously deleted for PEP4, PRB1 and PRC1, had a >5-fold stabilization of PtdIns(3)P (Figure 3). Vps34p PtdIns 3-kinase activity was not affected in the pep4Δ/prbΔ/prc1Δ strain (data not shown). This suggests that PtdIns(3)P turnover occurs in a hydrolase-dependent manner and that either PrA, PrB or CPY, or a combination of these hydrolases, participate in the activation of an enzyme(s) involved in PtdIns(3)P turnover. Given the role of PrA and PrB in the activation of vacuolar enzymes, it is likely that PrA and/or PrB, but not hydrolases like CPY or carboxypeptidase S (CPS) (Spormann et al., 1991), would be important to the maturation of PtdIns(3)P turnover proteins at the vacuole. Consistent with this, we found that PtdIns(3)P levels also increased 3.5-fold in TVY1 (pep4Δ) and 4-fold in TVY2 (prbΔ) cells lacking only PrA or PrB, respectively. This demonstrates that both PrA and PrB activity are important factors in PtdIns(3)P turnover. Deletion of PRC1 or CPS1 did not result in a change in PtdIns(3)P levels demonstrating that not all protease activities are required for the turnover of PtdIns(3)P. When we extend our analysis to fraction 75, wild-type and prb1Δ cells also exhibited peaks of similar magnitude (∼400 c.p.m.) which co-eluted with [3H]PtdIns(4,5)P2 standards (data not shown). Therefore, decreases in vacuolar hydrolase activity specifically affect PtdIns(3)P since neither PtdIns(4)P (Figure 3) or PtdIns(4,5)P2 levels were influenced in prb1Δ cells. Figure 3.Cellular PtdIns(3)P levels increase in strains that are deficient in vacuolar hydrolase activity. The indicated strains, including TVY6 (prc1Δ) and CCY241 (cps1Δ), were labeled for 12 h with myo-[2-3H]inositol, lysed and extracted in chloroform/methanol. Lipids were deacylated and separated by HPLC. Representative data from several experiments are shown. PtdIns(3)P and PtdIns(4)P are indicated. Download figure Download PowerPoint We also tested a strain deleted for the VMA4 gene, which encodes a component of the vacuolar ATPase (Ho et al., 1993). Mutation of VMA4 inactivates the vacuolar ATPase and prevents vacuole acidification (Morano and Klionsky, 1994). Studies indicate that certain vacuolar enzymes, including PrA, are sensitive to increases in pH (Sorensen et al., 1994), and thus are 30–40% less active (Morano and Klionsky, 1994). Consistent with this moderate effect on vacuolar hydrolase activity, deletion of VMA4 caused a 2.5-fold stabilization of PtdIns(3)P. Together, these results indicate that PtdIns(3)P is delivered to the vacuole and turned over by a PrA- and PrB-dependent pathway. Because PtdIns(3)P is synthesized in the cytoplasmic leaflet of the membrane (Herman et al., 1991), and PrA and PrB are active in the lumen of the vacuole, our data suggest that it might be necessary to transfer PtdIns(3)P to the interior of the vacuole in order for it to be exposed to and turned over by active hydrolases. A critical step in shifting lipids of the cytoplasmic leaflet to the lumen of an organelle may entail the incorporation of such lipids into membrane invaginations. Unlike wild-type cell vacuoles, which appear as dark, electron-dense compartments when viewed by electron microscopy (Banta et al., 1988), an interesting feature of the vacuoles in vma4Δ cells is that they are electron-transparent. This revealed the presence of intravacuolar vesicles (Figure 4A and B). These vesicles are uniform in appearance, being 40–50 nm in diameter and having a single limiting membrane. Membrane tubules, which when cross-sectioned should sometimes appear as elongated structures, were not observed in the vacuoles of vma4Δ cells prepared by this methodology (see Materials and methods). Like wild-type cells, prb1Δ cells exhibited electron-dense vacuoles that mask their intralumenal contents (data not shown). It is possible that these vesicles are derived from vacuolar invaginations or endosomal multivesicular bodies (MVBs) which are thought to fuse with lysosomal/vacuolar compartments, depositing vesicles into the lumen of the lysosome/vacuole (Futter et al., 1996; Cowles et al., 1997b). Interestingly, the vam3tsf mutant stabilized PtdIns(3)P (Figure 1B) and also accumulates prevacuolar MVBs (Darsow et al., 1997). If PtdIns(3)P is a component of these vesicles then PtdIns(3)P, together with these vesicles, could be degraded within the lumen of the vacuole (see Discussion). Figure 4.Electron microscopic analysis of vma4Δ mutant cells reveals intravacuolar vesicles. vma4Δ mutant cells were grown to mid-log phase, fixed and visualized by electron microscopy. Vesicles are indicated by arrowheads. (A) Whole-cell image (bar = 0.5 μm). (B) High magnification image of vacuolar vesicles (bar = 0.1 μm). V, vacuole; M, mitochondria; N, nucleus. Download figure Download PowerPoint A major pool of PtdIns(3)P is turned over at the vacuole To address whether the vacuole represents a major site for PtdIns(3)P turnover or whether the vacuole is responsible for degrading only a small portion of the total pool of PtdIns(3)P produced by Vps34p, PtdIns(3)P levels were measured in vps34tsf/vam3tsf double-mutant cells. At the restrictive temperature, both production of PtdIns(3)P and the transport of PtdIns(3)P to its site of turnover at the vacuole is expected to be blocked, revealing the contribution of the vacuolar degradative pathway to total PtdIns(3)P turnover. Accordingly, PtdIns(3)P levels were measured in vps34tsf, vam3tsf and in AWY1 (vps34tsf/vam3tsf) double-mutant cells after shifting these strains to the nonpermissive temperature for 0, 10 and 20 min. At the permissive temperature of 26°C, vps34tsf cells, vam3tsf cells and the vps34tsf/vam3tsf double-mutant all had relatively normal PtdIns(3)P levels (Figure 5, 0 min). As previously observed, cells expressing the vps34tsf allele exhibited ∼25% decreases in PtdIns(3)P, even at the permissive temperature (Stack et al., 1995). After a brief, 10-min shift to the nonpermissive temperature of 38°C, there was a 2-fold decrease in PtdIns(3)P levels in vps34tsf cells while vam3tsf cells exhibited a 1.7-fold increase in PtdIns(3)P (Figure 5, 10 min). Thus, significant decreases in PtdIns(3)P were observed within 10 min after shifting the vps34tsf strain to the nonpermissive temperature, suggesting that PtdIns(3)P turnover and the maturation of precursor CPY (half-life, 6–8 min) occur at similar rates (Klionsky et al., 1988). PtdIns(4)P levels were not affected upon temperature shift in this experiment (data not shown). However, even after 20 min at 38°C, PtdIns(3)P levels remained nearly constant in the vps34tsf/vam3tsf double-mutant cells (Figure 5, 20 min). The same was true after 40 min at the nonpermissive temperature, the longest temperature-shift carried out (data not shown). Together, these data indicate that upon inactivation of the Vps34 PtdIns 3-kinase, the vam3tsf mutant transport block stabilized the bulk of PtdIns(3)P. The vacuole, therefore, represents a major site for PtdIns(3)P turnover in yeast. Figure 5.PtdIns(3)P levels are stabilized in cells that are simultaneously compromised in PtdIns 3-kinase activity and PtdIns(3)P transport to the vacuole. PtdIns(3)P levels were assayed in vps34tsf cells, vam3tsf cells and in vps34tsf/vam3tsf double mutants. vps34tsf, vam3tsf and vps34tsf/vam3tsf strains were labeled for 12 h with myo-[2-3H]inositol at the permissive temperature, 26°C, and shifted to the nonpermissive temperature of 38°C for 0, 10 or 20 min. Lipids extracted from cells after lysis were deacylated and resolved by HPLC. Levels of deacylated PtdIns(3)P are shown as a function of time at the nonpermissive temperature. Download figure Download PowerPoint Vacuolar transport of PtdIns(3)P requires the CPY sorting pathway but not the alkaline phosphatase, late secretory or early endocytic pathways Multiple transport pathways to the vacuole have been documented. The CPY pathway traffics hydrolases such as CPY and CPS to the vacuole but not alkaline phosphatase (ALP), which instead relies on a distinct ALP pathway (Cowles et al., 1997b; Piper et al., 1997). The Ste6 protein (plasma membrane a-factor transporter), in contrast, follows the secretory pathway to the plasma membrane and then the endocytic pathway to the vacuole where it is degraded (Kolling and Hollenberg, 1994). The subset of class B genes represented by VAM3 and YPT7 are required for a late stage along many of these transport routes (Wichmann et al., 1992; Darsow et al., 1997). Therefore, it is possible that PtdIns(3)P traffics via the CPY pathway, the ALP pathway, the endocytic pathway or a combination of these pathways to the vacuole. To determine more precisely which of the Vam3p- and Ypt7p-dependent transport pathways are required to deliver PtdIns(3)P to the vacuole, we investigated whether specific blocks in a subset of these membrane-trafficking pathways could influence endogenous PtdIns(3)P levels. Vps4p, a class E Vps protein which encodes an ATPase, mediates trafficking along the CPY pathway and a late step of endocytosis but not the transport of ALP to the vacuole (Munn and Riezman, 1994; Babst et al., 1997). Vps4p activity is required for endosome function, and shifting the vps4ts strain to the nonpermissive temperature results in the accumulation of endosomal structures which contain CPY (Babst et al., 1997). When the vps4ts strain was labeled with myo-[2-3H]inositol at the permissive temperature and shifted, for the final 15 min of labeling, to the nonpermissive temperature, we observed a 1.4-fold increase in PtdIns(3)P (Figure 6). This suggests that at least a pool of PtdIns(3)P transits a Vps4p endosome compartment and that the CPY and/or the endocytic pathway are required to transport PtdIns(3)P to the vacuole. To determine if PtdIns(3)P is secreted to the plasma membrane and then endocytosed to the vacuole, we assayed PtdIns(3)P levels in sec1 and end4 temperature-sensitive strains. When shifted to the nonpermissive temperature, sec1 and end4 mutants block the late secretory and early endocytic pathways, respectively (Novick et al., 1980; Raths et al., 1993). PtdIns(3)P levels did not increase in either of these strains after a 15 min shift to 38°C (Figure 6), suggesting that PtdIns(3)P does not follow this route to the vacuole. Inactivation of the AP-3 adaptor complex through the deletion of APM3 blocks transport of ALP to the vacuole but does not block transport via the CPY pathway (Cowles et al., 1997a). PtdIns(3)P levels did not change in the apm3Δ1 strain (Figure 6). Collectively, these results indicate that a significant pool of PtdIns(3)P is trafficked by the CPY pathway from a CPY-containing endosome to the vacuole. In contrast, PtdIns(3)P transport does not depend on the ALP, late secretory or early endocytic pathways. This is consistent with the known functional role of Vps34p in the CPY pathway but not the ALP pathway (Stack et al., 1995). Figure 6.Cellular PtdIns(3)P levels in vps4ts, end4ts, sec1ts and apm3Δ1 strains. Two samples of wild-type, vps4ts, RH268-1C (end4ts) and sec1ts cells were labeled for 12 h with myo-[2-3H]inositol at 26°C. One sample of each strain was then shifted to 38°C for the final 15 min of labeling while the other sample was maintained at 26°C. GOY3 (apm3Δ1) cells were also labeled for 12 h at 26°C. All strains were then subjected to glass bead lysis in the presence of acidified chloroform/methanol. The extracted lipids were deacylated and analyzed by HPLC. Representative PtdIns(3)P counts recovered in multiple experiments are indicated. Download figure Download PowerPoint Vps34p PtdIns 3-kinase activity is required for efficient delivery of endocytic cargo to the vacuole While the PtdIns 3-kinase activity of Vps34p is known to be critical for the trafficking of newly synthesized hydrolases from the Golgi to the vacuole (Herman and Emr, 1990; Stack et al., 1995), the specific site(s) of PtdIns(3)P function in the vacuolar transport pathway has not been defined. The data presented here indicate that a significant pool of PtdIns(3)P is trafficked from endosomal compartments to the vacuole, making PtdIns(3)P, at least briefly, a cargo component of endosomal membranes. These results raise the possibility that PtdIns(3)P plays a role in endosome function. To address the possibility that Vps34p activity is required for endosome-to-vacuole trafficking, the endocytosis of FM4-64 and Ste6p was monitored in the vps34tsf strain after shifting to the nonpermissive temperature. FM4-64 is a fluorescent lipophilic molecule which intercalates into the plasma membrane when added exogenously to yeast cells (Vida and Emr, 1995). The endocytic progress of FM4-64 to the vacuole can then be monitored by fluorescence microscopy (Wendland et al., 1996). Wild-type and vps34tsf cells were grown at the permissive temperature (26°C) and pre-shifted for 10 min to the nonpermissive temperature of 37°C. The cells were then pulse-labeled with FM4-64 for 8 min and the trafficking of FM4-64 to the vacuole monitored at various chase times in FM4-64-free media. After 10 min of chase, the bulk of FM4-64 reached the vacuole in wild-type cells while vps34tsf cells exhibited limited vacuole staining and the accumulation of FM4-64 in punctate spots immediately peripheral to the vacuole (Fig
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