ATG23, a novel gene required for maturation of proaminopeptidase I, but not for autophagy

In rich media proaminopeptidase I is targeted to the vacuole via the Cvt pathway and during starvation via autophagy. We here identify Atg23 (Ylr431c), a protein of so far unknown function, as a novel component essential for proaminopeptidase I maturation under non-starvation conditions. Maturation of proaminopeptidase I takes place in starved atg23Delta cells. Selective vacuolar targeting of the autophagosomal marker GFP-Aut7 and the accumulation of autophagic bodies during starvation in the presence of phenylmethylsulfonyl fluoride suggest that autophagy occurs in atg23Delta cells but at a reduced rate. In atg23Delta cells mature vacuolar carboxypeptidase Y is present and accumulation of quinacrine suggests no significant defect in vacuolar acidification. Furthermore, growth of atg23Delta cells on nitrocellulose detects no significant secretion of carboxypeptidase Y.     

Yeast Vps55p,a functional homolog of human obesity receptor gene-related protein,is involved in late endosome to vacuole trafficking

The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae

Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.     

Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy

Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.     

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.     

Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.     

Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.     

Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to plasma membrane transporters for efflux?

In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein-sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein-sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.     

A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Vrl1 relies on its VPS9‐domain to play a role in autophagy in Saccharomyces cerevisiae

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9‐domain‐containing protein, V AR P‐l ike 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9‐domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9‐domain‐containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9‐domain‐containing proteins, Vps9, Muk1, and Vrl1, all co‐localized with Atg8 on autophagosomes in cells blocked in any late step of starvation‐induced autophagy, with Vrl1 most often co‐localizing with Atg8. A small portion (<25%) of these VPS9‐domain‐containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy‐dependent and ‐independent routes.     

Investigation of peroxisome biogenesis revealed possible new roles for autophagy components

Precursor aminopeptidase I oligomerizes in the cytosol and is imported into the vacuole as a dodecamer via the cytoplasm-to-vacuole targeting (Cvt) pathway or autophagy. However, this is not the only example for the import of oligomeric protein complexes into an organelle. During peroxisome biogenesis folded and oligomeric proteins can be imported into the lumen of the organelle. The mechanism of this transport is still unknown. In this article, we point out mechanistic parallels between peroxisome biogenesis and the Cvt pathway or autophagy. Furthermore, we summarize our recently published investigation on a possible overlap between these pathways. Our investigation revealed new insights into autophagy and the Cvt pathway and possible new functions of Cvt4p, Cvt8p and Atg14p in organelle biogenesis or stability.(1).     

A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery.

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.     

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.     

Characterization of phosphatidylserine transport to the locus of phosphatidylserine decarboxylase 2 in permeabilized yeast

In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mitochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidylethanolamine (PtdEtn). In strains with a psd1Delta allele for the mitochondrial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can serve as an indicator of lipid transport to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole. We have followed the metabolism of [(3)H]serine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1Delta yeast. The permeabilized cells synthesize (3)H-PtdSer and, after a 20-min lag, decarboxylate it to form [(3)H]PtdEtn. Formation of [(3)H]PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing PtdSer synthesis. PtdSer transport can be resolved into a two-component system using washed, permeabilized psd1Delta cells as donors and membranes isolated by ultracentrifugation as acceptors. With this system, the transport-dependent decarboxylation of nascent PtdSer is dependent upon the concentration of acceptor membranes, requires Mn(2+) but not nucleotides, and is inhibited by EDTA. High speed membranes isolated from a previously identified PtdSer transport mutant, pstB2, contain normal Psd2p activity but fail to reconstitute PtdSer transport and decarboxylation. Reconstitution with permutations of wild type and pstB2Delta donors and acceptors identifies the site of the mutant defect as the acceptor side of the transport reaction.     

VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins.

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.     

Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11Delta and atg19Delta strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8Delta atg11Delta double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.     

Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is independent of its ability to form hexagonal phase structures

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity. When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine. In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.