Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Degradation of Lipid Vesicles in the Yeast Vacuole Requires Function of Cvt17, a Putative Lipase

Degradation of Lipid Vesicles in the Yeast Vacuole Requires Function of Cvt17, a Putative Lipase THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 1, Issue of January 19, pp. 2083–2087, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Degradation of Lipid Vesicles in the Yeast Vacuole Requires Function of Cvt17, a Putative Lipase* Received for publication, October 18, 2000, and in revised form, November 7, 2000 Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.C000739200 Sarah A. Teter‡, Kimberly P. Eggerton‡, Sidney V. Scott§, John Kim‡, April M. Fischer‡, and Daniel J. Klionsky‡¶ From the ‡University of Michigan, Department of Biology, Ann Arbor, Michigan 48109 and the §Section of Microbiology, University of California, Davis, California 95616 The vacuole/lysosome serves an essential role in al- cytoplasm to the vacuolar lumen. Cvt and autophagy employ many of the same molecular components and are mechanisti- lowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are cally related (3– 6). Both pathways involve the formation of key proteins in this process. In Saccharyomces cerevi- double-membrane cytosolic vesicles, sequestering either pre- siae, some resident vacuolar hydrolases are delivered by cursor aminopeptidase I (prAPI) specifically, or in the case of the cytoplasm to vacuole targeting (Cvt) pathway, which autophagy, also enveloping bulk cytosol in a nonselective man- shares mechanistic features with autophagy. Autophagy ner. Fusion of these vesicles with the vacuole results in the is a degradative pathway that is used to degrade and release of single-membrane subvacuolar vesicles within the recycle cellular components under starvation condi- lumen. These pathways require a mechanism for specific lysis tions. Both the Cvt pathway and autophagy employ dou- of the internalized vesicles, so that vesicle cargo can be released ble-membrane cytosolic vesicles to deliver cargo to the into the vacuole lumen, and further require a mechanism for vacuole. As a result, these pathways share a common degradation of vesicle lipids. terminal step, the degradation of subvacuolar vesicles. To understand the molecular basis of these import and deg- We have identified a protein, Cvt17, which is essential radation pathways, we carried out a genetic screen for mutants for this membrane lytic event. Cvt17 is a membrane defective in delivery of prAPI to the vacuole. We isolated a glycoprotein that contains a motif conserved in ester- series of mutants, cvt, which accumulate the precursor form of ases and lipases. The active-site serine of this motif is API. The cvt mutants overlap with mutants defective in auto- required for subvacuolar vesicle lysis. This is the first phagy. The majority of these mutants are blocked at a stage characterization of a putative lipase implicated in vac- involving formation of the sequestering vesicle (reviewed in uolar function in yeast. Ref. 1). One mutant, cvt17, was found to be blocked in the breakdown of subvacuolar vesicles, suggesting that Cvt17 acts at a late stage of the import process (6). We report in this paper One fundamental role of the yeast vacuole is in the recycling the cloning of the gene encoding CVT17. Immunological and of biological macromolecules. The vacuole, like the lysosome in biochemical studies demonstrate that Cvt17 is a glycosylated, animal cells, is the primary site of degradation. Our under- integral membrane protein that transits through the secretory standing of the hydrolytic vacuolar enzymes that serve in pro- pathway. Cvt17 contains a domain that is conserved among tein turnover is well advanced. Progress has also been made in lipases of the a/b-hydrolase-fold superfamily. Mutation of the elucidating mechanisms that deliver the substrates of these putative lipase active site abolishes Cvt17 function in the Cvt vacuolar hydrolases. While research has focused on the biosyn- and autophagy pathways, suggesting that lipase activity is thesis and function of the vacuolar proteases, little is known critical to the role of this protein in these import processes. about how lipids are recycled in this organelle, and a lipase that functions in membrane recycling has not been identified. EXPERIMENTAL PROCEDURES Nearly all vacuolar/lysosomal delivery pathways involve Strains and Media—Wild type yeast strain SEY6210, was described packaging of cargo within membrane-enclosed transport com- previously (7), as were the mutant strains THY32 (cvt17-1; Ref. 3), partments. Because the vacuole/lysosome serves as the final MGY101 (apg5D::LEU2; Ref. 8), TVY1 (pep4D::LEU2; Ref. 9), WSY99 destination for these numerous vesicle-mediated transport (ypt7D::HIS3; Ref. 10), and NNY20 (apg1D::LEU2; Ref. 11). Strain WPHYD7 SEY6210 aut7D::LEU2 will be described elsewhere. The pathways, the issue of how membranes reaching the vacuole cvt17D strain is described below. Yeast cells were grown as described are recycled is an important one. Macroautophagy is the major previously (12). degradative process in eukaryotes and is essential during star- Materials—We followed standard procedures to prepare antiserum vation conditions (1). In yeast, autophagy overlaps with a bio- to Cvt17, using synthetic peptides corresponding to amino acids 131– synthetic process, the Cvt pathway, that delivers the hydrolase 152 and 295–312 (Multiple Peptide Systems, San Diego, CA). Addi- aminopeptidase I (API ; Ref. 2) from its site of synthesis in the tional antisera were described previously (13–15). The copper-inducible promoter-based plasmid pCu426 was from Dr. Dennis J. Thiele (Uni- * This work was supported by Public Health Service Grant GM53396 from the National Institutes of Health (to D. J. K.). The costs of publi- sor API; Cvt, cytoplasm to vacuole targeting; Endo H, endoglycosidase cation of this article were defrayed in part by the payment of page H; ER, endoplasmic reticulum; GFP, green fluorescent protein; PMSF, charges. This article must therefore be hereby marked “advertisement” phenylmethylsulfonyl fluoride; SD-N, synthetic minimal medium lack- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ing nitrogen; ORF, open reading frame; SGD, Saccharomyces Genome ¶ To whom correspondence should be addressed. Tel.: 734-615-6556; Database; PCR, polymerase chain reaction. Fax: 734-647-0884; E-mail: [email protected]. Kim, J., Huang, W.-P., and Klionsky, D. J. (2001) J. Cell Biol. 152, The abbreviations used are: API, aminopeptidase I; prAPI, precur- in press This paper is available on line at http://www.jbc.org 2083 This is an Open Access article under the CC BY license. 2084 Cvt17, a Putative Lipase Required for Cvt and Autophagy versity of Michigan; Ref. 16). Other reagents are identical to those described previously (12, 17). Cloning, Disruption, and Mutagenesis of CVT17—The CVT17 gene was cloned by transforming the cvt17-1 strain with a plasmid genomic library and screening for complementation of the starvation-sensitive phenotype on plates containing phloxine B (18). A secondary screen for prAPI accumulation was carried out as described previously (2). Sub- cloning of a complementing plasmid identified YCR068w as the com- plementing CVT17 ORF. The cvt17D strain (KTYD17) was made by inserting the LEU2 gene into the CVT17 ORF of strain SEY6210. At the time of this analysis, the Saccharomyces Genome Database (SGD) se- quence had shown YCR068w as a 1.289-kilobase coding sequence. We amplified this region and subcloned it into pCu426 using PCR (all primer sequences available upon request). This construct (pCuCVT17DC(426)) failed to complement the prAPI defect of cvt17D. The construct was sequenced, and comparison to the SGD sequence revealed a single cytosine insertion in the PCR amplified gene, at nucleotide 956 of the ORF. Sequencing of the original genomic plasmid revealed an identical sequence to the PCR product, suggesting an error in the SGD sequence. A recent revision of the chromosome III sequence has corrected this error. The rectified CVT17 sequence shifts the frame of the coding se- quence, increasing the length of the encoded protein. The longer, full- length coding sequence was amplified by PCR and subcloned into FIG.1. The cvt17D strain accumulates Cvt bodies within the pCu426 to generate pCuCVT17(426). For analysis of Cvt17 expression vacuole. A, precursor API is enclosed within a membrane compart- driven by the native promoter, CVT17 was amplified by PCR and cloned ment in the cvt17D strain. Osmotically lysed spheroplasts (T) were into pRS416 and pRS426, with inclusion of 0.5 kilobase before the start separated into low speed supernatant (S) and pellet (P) fractions by a codon and 0.432 kilobase after the stop codon. A PCR-based overlap- 5,000 3 g centrifugation. P fractions were resuspended in lysis buffer extension strategy was used in site-directed mutagenesis of CVT17. alone or treated with proteinase K 6 Triton X-100. B, the Cvt vesicle Serine 332 was converted to alanine by changing the T at nucleotide 994 marker protein GFPAut7 accumulates in subvacuolar vesicles in to G. cvt17D. pep4D and cvt17D cells harboring the pCuGFPAUT7(416) plas- Other Procedures—Cell fractionation, immunoprecipitation of radio- mid were grown to mid-log phase. GFPAut7 expression was induced labeled proteins, nitrogen starvation sensitivity, prAPI protease sensi- with 10 mM CuSO for 3 h. Vacuoles were labeled with FM 4-64. tivity, and reversal in SD-N were performed as described previously (8, 19). For endoglycosidase H (Endo H) treatment, cells were labeled for 10 a pelletable fraction in cvt17D, as it was in the apg5D and ypt7D min with no chase. Immunoprecipitated proteins were resuspended in mutant strains (Fig. 1A). In the apg5D strain, prAPI was sen- 0.1% SDS, 50 mM sodium citrate, pH 5.5, 1 mM PMSF. After boiling for 7 min, an equal volume of 0.1% SDS and 0.1 M b-mercaptoethanol was sitive to exogenous protease, even in the absence of detergent, added, then boiled for 7 min. Denatured proteins were incubated over- consistent with a block in vesicle formation (8). In contrast, night at 37° C in the presence of 50 mM sodium citrate, pH 5.5, 16 mM ypt7D and cvt17D cells contained prAPI that was not degraded b-mercaptoethanol, 0.03% SDS, 5 mM PMSF, and 2 units of Endo H. A to the mature form in the presence of exogenously added pro- second immunoprecipitation recovered the Cvt17 proteins. teinase K unless detergent was also added, indicating that Fluorescence Microscopy—Construction of the GFPAut7 fusion pro- prAPI was within a membrane enclosed compartment (Fig. 1A; tein and FM 4-64 labeling of cells are described elsewhere. Cells were examined using a Nikon Eclipse E-800 fluorescence microscope. Images Ref. 21). These data, coupled with the vesicle accumulation were captured by a Hamamatsu C4742–98 digital camera. phenotype of cvt17-1, suggest that prAPI was accumulating within subvacuolar Cvt vesicles in the cvt17D strain. RESULTS To assess whether the vesicles accumulating in cvt17D are Cvt17 Is Required for Lysis of Subvacuolar Cvt Bodies and indeed Cvt bodies, we examined whether a Cvt vesicle marker Autophagic Bodies—Previous analysis of cvt17-1 by electron accumulated within subvacuolar vesicles during vegetative microscopy suggested a defect in the breakdown of subvacu- growth conditions. Aut7 is a component that is required for Cvt olar, prAPI-containing vesicles, called Cvt bodies (3, 6). We vesicle formation and is itself enclosed within Cvt vesicles (22). isolated the CVT17 gene and disrupted the chromosomal locus We have recently demonstrated the utility of a GFPAut7 con- to generate a cvt17D strain (see “Experimental Procedures”). struct as a marker for following the formation and movement of The null phenotype was accumulation of prAPI (Fig. 1A), as Cvt vesicles in vivo. To visualize the vacuole membrane, we seen previously with the cvt17-1 mutant. used FM 4-64, a lipophilic red dye. In cvt17D, GFPAut7 is To determine the site of action of Cvt17, we analyzed the localized to Cvt bodies, seen as punctate structures accumulat- state of precursor API in cvt17D. Yeast cells were converted to ing within FM 4-64-labeled vacuoles (Fig. 1B, top panels). Some spheroplasts and subjected to osmotic lysis. Addition of exoge- GFPAut7 punctate staining was also observed outside the vac- nous protease allowed us to determine whether the accumu- uole. Mutants in the PEP4 gene, encoding vacuolar proteinase lated prAPI was blocked at a stage prior to Cvt vesicle forma- A, accumulate subvacuolar vesicles, including Cvt bodies by tion/completion (protease-sensitive) or at a point following electron microscopy (5). Consistent with these previous obser- enclosure but prior to fusion with the vacuole or vesicle break- vations, GFPAut7-labeled Cvt bodies also accumulated in the down (protease-protected). As controls, we examined prAPI vacuoles of a pep4D strain (Fig. 1B, bottom panels) but did not protease sensitivity in two strains that are defective in prAPI accumulate in the vacuoles of wild type yeast (data not shown). import. Apg5 is part of a novel protein conjugation complex The FM 4-64 vacuolar staining in cvt17D differed from that in that is required for both the Cvt and autophagy pathways (20). other strains, including pep4D; we observed a relative increase The apg5D strain is blocked in the completion of Cvt vesicles in the number of FM 4-64-labeled compartments per cell in the and accumulates membrane-associated, but protease-sensitive, cvt17D strain (Fig. 1B). While we do not know the molecular prAPI (8). Ypt7 is a rab GTPase that is required for fusion of basis for the change in vacuolar morphology, the microscopy Cvt vesicles and autophagosomes with the vacuole (1). The data provide direct evidence that cvt17D accumulates Cvt bod- ypt7D mutant accumulates prAPI in a protease-resistant state ies within the vacuole. within completed cytosolic vesicles. Precursor API was found in While many molecular components are shared between the Cvt17, a Putative Lipase Required for Cvt and Autophagy 2085 FIG.2. The cvt17D strain is defective in autophagy. A, log phase cultures grown in 1% yeast extract, 2% peptone, 2% glucose (YPD) were shifted to medium lacking nitrogen (SD-N). Viability was determined by plating aliquots on YPD plates. B, indicated strains were shifted to SD-N as in A, with aliquots taken at indicated times. Crude lysates were subjected to immunoblot analysis with anti-API antiserum. FIG.3. The nucleophilic serine of a putative lipase domain is Cvt and autophagy pathways, some cvt mutants are not defec- required for Cvt17 function. A, wild type yeast or strain cvt17D with the indicated CVT17-containing plasmids were grown to mid-log stage. tive in autophagy (3, 12). To test whether cvt17D has a defect in Western blots with anti-API antiserum are shown. Plasmids: library, autophagy, we examined whether the strain can survive complementing library plasmid; DC-term. 2m, truncated CVT17 gene growth in nitrogen-depleted media. Because transport to the with the last 87 residues deleted cloned in pCu426; 2m and CEN, vacuole by autophagy is the primary mode for degradation of full-length CVT17 gene in pRS426 and pRS416, respectively; S332A 2m and S332A CEN, Cvt17 with Ser to Ala mutation in the putative cytoplasmic constituents under starvation conditions, the proc- catalytic site cloned in pRS426 and pRS416, respectively. B, sequence ess is essential for viability during nutrient limitation. Wild alignments of Cvt17 and related fungal proteins (see “Results” for type cells can withstand nutrient deprivation for long periods of details). Gaps in the alignment (-), fully conserved residues (*), and time, while autophagy mutants such as apg1D or protease- residues with strong (:) and weaker (.) similarity are indicated. Putative catalytic triad residues (S-D-H) are indicated by outlined boxes; only deficient strains such as pep4D show reduced viability follow- one of the conserved Asp residues is expected to be active. The gray box ing a shift from growth in nutrient-rich medium to nitrogen- indicates the sequence that matches the consensus lipase pattern. deficient medium (Fig. 2A). Like apg1D and pep4D, the cvt17D ClustalX software was used to align sequences. strain was starvation-sensitive. Growing yeast cells transport prAPI to the vacuole via Cvt vesicles, while under nutrient deprivation, prAPI can be trans- kDa, rather than the 49.9 kDa predicted in the original data ported by autophagosomes (4, 5). If cvt17D is defective in lysis base. The full-length gene is able to complement the cvt17D of autophagic bodies, as its starvation sensitivity suggests, we strain, as shown by the accumulation of mAPI (Fig. 3A, 2m and should see a block in prAPI maturation not only in rich media CEN). Importantly, the frameshift occurred just 59 to a consen- but also under starvation conditions. Indeed, both the Cvt and sus site that is conserved in lipases and esterases (EC 3.1.1.3). autophagic routes of delivery are blocked, as indicated by anal- The presence of this motif was revealed by a BLOCK search ysis of API following a shift to SD-N medium (Fig. 2B). Auto- using the full-length, correct Cvt17 sequence. The sequence phagy mutants display differential blocks in API import under from amino acid 324 to 338 matches the consensus pattern of nutrient-rich versus starvation conditions. For example, mu- serine-active lipases (PROSITE reference PS00120; [LIV]-X- tants such as aut7D that do not have a complete defect in [LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]; Fig. 3B). A autophagy accumulate prAPI in nutrient-rich media but are BLAST search identified related proteins in several distinct TM able to mature prAPI under starvation conditions (Ref. 19; Fig. fungi, including Candida albicans (GenBank accession num- 2B). In contrast, strains that have more severe autophagy ber AL033391; 43% identity), as well as the fission yeast Schiz- TM defects, including apg1D, accumulate prAPI even after pro- osaccharomyces pombe (GenBank accession number Z99753; longed growth in SD-N. The cvt17D strain did not exhibit a 48% identity) and the tomato pathogen Cladosporium fulvum TM rescue of the prAPI processing defect when cells were shifted to (GenBank accession number Y14554; 38% identity (23)). SD-N, indicating that it is defective in autophagy as well as the Alignments of the partial sequences over an area of significant Cvt pathway (Fig. 2B). From these results we conclude that homology are shown in Fig. 3B. Cvt17 is most similar to the Cvt17 is involved in the lysis of autophagic bodies, as well as subfamily of fungal lipases within the larger a/b-hydrolase-fold Cvt bodies. superfamily (24, 25). Triglcyeride lipases are lipolytic enzymes Putative Serine Nucleophile in a Consensus Lipase Active that hydrolyze the ester bond of triglycerides. In addition to the Site Is Required for Cvt17 Function—Our original analysis of conserved serine within the active site consensus motif, a his- CVT17 revealed an SGD error in the coding region of this ORF tidine residue and an aspartic acid residue act in hydrolysis in (see “Experimental Procedures”). We discovered that the pre- a charge-relay system (26). We found two well conserved as- dicted coding sequence that was given in the data base at that partic acid residues in Cvt17 and other related fungal genes as time did not complement the prAPI accumulation phenotype in well as a conserved histidine (Fig. 3B). cvt17D (Fig. 3A, DC-term. 2 m). Sequencing revealed the pres- To determine whether the serine of the putative lipase do- ence of an extra cytosine base in the gene, at nucleotide 956 of main is required for the function of Cvt17, we mutated the the ORF. This insertion shifts the predicted reading frame, serine at position 332 to an alanine residue. Western blot resulting in a protein with a predicted molecular mass of 58.5 analysis of API in the cvt17D strain that contained the mutated 2086 Cvt17, a Putative Lipase Required for Cvt and Autophagy FIG.5. Cvt17 is a short-lived glycoprotein. A, cells were labeled for 10 min, followed by immunoprecipitation with antiserum to Cvt17. Immunoprecipitated proteins were mock treated or treated with Endo H. B, wild type cells were labeled for 10 min. Crude extracts from FIG.4. Cvt17-specific antiserum recognizes a 70-kDa mem- samples collected at the indicated chase times were immunoprecipi- brane protein. A, cells from cvt17D, wild type and cvt17D cells har- tated with Cvt17-specific antiserum. boring 2m wild type CVT17 and S332A mutant plasmids were radiola- beled for 5 min followed by a 10-min chase. Crude extracts were immunoprecipitated with antiserum to Cvt17. B, spheroplasts of wild vation that Cvt17 is glycosylated allows us to infer the mem- type cells were osmotically lysed, and the resultant lysate (T) was brane topology of the protein. All three putative glycosylation centrifuged at 13,000 3 g to yield supernatant (S13) and pellet (P13) sites are C-terminal to the transmembrane domain, suggesting fractions. S13 was further separated into high speed S100 and P100 that Cvt17 is a type II integral membrane protein. Upon inte- fractions by centrifugation at 100,000 3 g. Samples were subjected to immunoblot analysis with antisera to Cvt17, the vacuolar membrane- gration into the ER membrane, the N-terminal 13 amino acids associated protein Vac8, and the cytosolic protein phosphoglycerate would remain exposed to the cytosol, with the majority of the kinase (PGK). protein located in the lumen of the ER. The phenotype of the cvt17 mutant, accumulation of sub- gene (cvt17S332A) showed accumulation of the precursor form vacuolar vesicles, coupled with its characterization as a secre- of API, even when the mutant gene was expressed from a high tory pathway protein suggests that Cvt17 might function copy plasmid (Fig. 3A). In contrast, the wild type gene allowed within the vacuole lumen. Accordingly, we attempted to local- for prAPI maturation on both high and low copy plasmids. ize the Cvt17 protein. Subcellular fractionation based on veloc- Thus, Cvt17 cannot function to break down subvacuolar vesi- ity sedimentation indicated that Cvt17 was predominantly lo- cles and allow prAPI to be released into the vacuole without calized within a 13,000 3 g fraction (Fig. 4B). The P13 fraction this serine within the G-X-S-X-G lipase motif. contains various subcellular compartments, including the vac- Cvt17 Is a Glycosylated, Integral Membrane Protein—The uole. To directly determine whether Cvt17 was located within potential assignment of Cvt17 as a lipase required for the the vacuole, we purified vacuoles from yeast spheroplasts. degradation of subvacuolar vesicles led us to examine its bio- However, we were unable to detect Cvt17 in the vacuolar synthesis. To this end, we generated polyclonal antiserum fraction using this technique (data not shown). As an alterna- against the protein. Immunoprecipitation revealed a single tive approach, we fractionated cell lysates on an OptiPrep predominant band of ;70 kDa. This band was not detected in density gradient. Using this technique, in the presence of sub- the cvt17D strain, and expression of CVT17 from a high copy stantial amounts of protease inhibitors, we were able to detect plasmid greatly increased the abundance of this protein (Fig. small amounts of Cvt17 in fractions that contained the vacuo- 4A, 2m lanes), suggesting that the serum was specific for Cvt17. lar protein marker alkaline phosphatase (data not shown). We also immunoprecipitated Cvt17 from the strain harboring However, the majority of the Cvt17 was found in fractions that the mutant protein, which lacked the putative catalytic serine were distinct from the vacuole fractions. Thus, Cvt17 is found (cvt17S332A 2m). The altered protein was recovered in equiv- predominately in a membrane fraction residing outside the alent amounts to the wild type protein, indicating that the vacuole under steady-state conditions. single point mutation did not affect the stability of the protein. To examine the localization of Cvt17 in vivo, we engineered Analysis of the Cvt17 primary sequence indicates a stretch of GFP fusions with the protein at both the N and C termini. Both hydrophobic amino acids (residues 13–35) that might serve as constructs only partially complemented the Cvt trafficking de- a membrane anchor for the protein. Following osmotic lysis and fect of the cvt17D strain, as assessed by API Western blotting subcellular fractionation, Cvt17 was predominately localized to (data not shown). Furthermore, we were unable to visualize a 13,000 3 g pelletable fraction (Fig. 4B). We investigated strong fluorescent signals anywhere in cells containing these whether this localization was due to direct interaction with a GFP fusion constructs. An explanation for this lack of signal membrane by subjecting the P13 fraction to different wash might be an inherent instability of Cvt17. To examine stability conditions. Alkali extraction efficiently removed the peripheral of Cvt17 directly, we carried out a pulse-chase analysis. Yeast membrane protein Vma2 from the membrane but had no effect cells were labeled with [ S]methionine/cysteine and subjected on Cvt17 or the integral membrane protein Dpm1 (data not to a nonradioactive chase. At each time point, samples were shown). Treatment with the detergent Triton X-100 resulted in removed and immunoprecipitated. We found that Cvt17 was the solubilization of all three proteins. These results suggest unstable in wild type cells; the protein was degraded with a that Cvt17 is an integral membrane protein. half-life of ;45 min (Fig. 5B). The inherent instability of this The predicted molecular mass of Cvt17 is 58.5 kDa; however, protein made its localization problematic as much of it was the protein migrated with a molecular mass of 70 kDa following degraded during fractionation procedures. SDS-polyacrylamide gel electrophoresis (Fig. 4A). The Cvt17 DISCUSSION sequence has three predicted N-linked glycosylation sites. To determine whether the aberrant migration was due to glycosy- We have identified a protein that is required for the turnover lation, we treated immunoprecipitated Cvt17 with Endo H. of membrane vesicles in the vacuole of yeast. Sequence analysis Following treatment with the deglycosylating enzyme, the mo- of this protein reveals the presence of a domain found in lipases lecular mass of Cvt17 shifted to ;61 kDa (Fig. 5A). This change and esterases, and we have confirmed the importance of this in molecular mass correlates well with the predicted removal of domain by using site-directed mutagenesis of a putative active three N-linked carbohydrate side chains. This post-transla- site serine residue within the lipase motif. Phenotypic analysis tional modification reveals that Cvt17 must reside at least of yeast expressing the altered Cvt17 protein shows that the temporarily in the secretory pathway. Furthermore, the obser- protein is inactive in the Cvt pathway without this residue. The Cvt17, a Putative Lipase Required for Cvt and Autophagy 2087 cvt17D mutant is unable to degrade both Cvt bodies and auto- Cvt17 function, and specifically the function of a lipase active phagic bodies and is extremely sensitive to starvation (Fig. 2A). site domain, is required for the lysis of subvacuolar Cvt and The ability to degrade membranes that are released into the autophagic bodies. Characterization of this protein is an im- vacuole lumen is required for starvation survival. In the ab- portant first step in understanding vacuolar function in the sence of this degradative capacity, the cell is unable to break turnover of lipids and in the terminal steps of the Cvt and down lipid and, as a result, is unable to access cargo contained autophagic pathways. Continued analysis of Cvt17 will provide within autophagic bodies. The occurrence of genes with simi- important information about membrane recycling in the larity to CVT17 in other fungi suggests that this may be a vacuole. conserved protein in the kingdom. Acknowledgments—We thank Jacob Teter for technical assistance, The maintenance of membrane-bound vesicles in the cvt17D Bob Fuller for use of the fluorescence microscope, and E. J. Brace for strain suggests a role for the protein in degrading a phospho- help with microscopy. We thank Maria Hutchins for comments on the lipid bilayer. Sequence comparisons, however, place the protein manuscript and for technical assistance. in a class of hydrolytic enzymes that act primarily on triglyc- REFERENCES erides, not phospholipids. It will be necessary to perform bio- 1. Kim, J., and Klionsky, D. J. (2000) Annu. Rev. Biochem. 69, 303–342 chemical studies with the purified enzyme to investigate its 2. Harding, T. M., Morano, K. A., Scott, S. V., and Klionsky, D. J. (1995) J. Cell substrate specificity. To function properly, lipases must be Biol. 131, 591– 602 targeted to the appropriate substrates. The membrane source 3. Harding, T. M., Hefner-Gravink, A., Thumm, M., and Klionsky, D. J. (1996) J. Biol. Chem. 271, 17621–17624 of Cvt vesicles and autophagosomes is not known. If Cvt17 is 4. Scott, S. V., Hefner-Gravink, A., Morano, K. A., Noda, T., Ohsumi, Y., and incorporated into Cvt vesicles and autophagosomes, its topol- Klionsky, D. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12304 –12308 5. Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., and Ohsumi, Y. (1997) J. Cell ogy would place the Cvt17 active site on the outer surface of the Biol. 139, 1687–1695 Cvt and autophagic bodies. To prevent membrane degradation 6. Scott, S. V., Baba, M., Ohsumi, Y., and Klionsky, D. J. (1997) J. Cell Biol. 138, during transit, it is possible that Cvt17 becomes active only in 37– 44 7. Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. the vacuole. If this is the case, the lytic activity of this protein Biol. 8, 4936 – 4948 must be suppressed during transit. Some aspect of the vacuolar 8. George, M. D., Baba, M., Scott, S. V., Mizushima, N., Garrison, B. S., Ohsumi, Y., and Klionsky, D. J. (2000) Mol. Biol. Cell 11, 969 –982 milieu may then activate Cvt17 as is thought to be the case for 9. Gerhardt, B., Kordas, T. J., Thompson, C. M., Patel, P., and Vida, T. (1998) proteinase A. If Cvt17 acts directly to degrade subvacuolar J. Biol. Chem. 19, 15818 –15829 vesicles, it should reside within the vacuole. Subcellular frac- 10. Wurmser, A., and Emr, S. (1998) EMBO J. 17, 4930 – 4942 11. Matsuura, A., Tsukada, M., Wada, Y., and Ohsumi, Y. (1997) Gene (Amst.) 192, tionation did reveal a small population of vacuolar Cvt17 that 245–250 may play a primary role in directly degrading Cvt and auto- 12. Scott, S. V., Nice, D. C., III, Nau, J. J., Weisman, L. S., Kamada, Y., Keizer- Gunnink, I., Funakoshi, T., Veenhuis, M., Ohsumi, Y., and Klionsky, D. J. phagic bodies. Most of the protein, however, did not cofraction- (2000) J. Biol. Chem. 275, 25840 –25849 ate with this organelle. In agreement with these results, the 13. Klionsky, D. J., Cueva, R., and Yaver, D. S. (1992) J. Cell Biol. 119, 287–299 rapid Cvt17 degradation that we observed (Fig. 5B) was inde- 14. Tomashek, J. J., Sonnenburg, J. L., Artimovich, J. M., and Klionsky, D. J. (1996) J. Biol. Chem. 271, 10397–10404 pendent of the major vacuole proteases (data not shown). 15. Wang, Y. X., Catlett, N. L., and Weisman, L. S. (1998) J. Cell Biol. 140, The reasons for maintaining a sizable population of Cvt17 1063–1074 16. Labbe ´ , S., and Thiele, D. J. (1997) Methods Enzymol. 306, 145–153 outside the vacuole are as yet obscure. It is possible that Cvt17 17. Noda, T., Kim, J., Huang, W.-P., Baba, M., Tokunaga, C., Ohsumi, Y., and performs some additional unknown function outside the vacu- Klionsky, D. J. (2000) J. Cell Biol. 148, 465– 479 ole. Another possibility is that Cvt17 acts exclusively outside 18. Tsukada, M., and Ohsumi, Y. (1993) FEBS Lett. 333, 169 –174 19. Abeliovich, H., Dunn, W. A., Jr., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. the vacuole and only indirectly influences the lytic competence 151, 1025–1033 of subvacuolar vesicles; Cvt17 might act as a lipase to specifi- 20. Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., and Ohsumi, Y. (1998) Nature 395, 395–398 cally alter the lipid composition of Cvt vesicles and autophago- 21. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., and Klionsky, D. J. (1999) somes as they form. Specific lipid composition might be a re- Mol. Biol. Cell 10, 1337–1351 quirement for the subsequent lysis of Cvt bodies and 22. Huang, W.-P., Scott, S. V., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. 275, 5845–5851 autophagic bodies. Lipids could even provide the molecular 23. Coleman, M., Henricot, B., Arnau, J., and Oliver, R. P. (1997) Mol. Plant basis for recognition within the vacuole lumen, allowing an Microbe Interact. 10, 1106 –1109 24. Cousin, X., Hotelier, T., Giles, K., Toutant, J. P., and Chatonnet, A. (1998) alternate vacuolar lipase to distinguish between subvacuolar Nucleic Acids Res. 26, 226 –228 vesicles destined for degradation and the bounding vacuolar 25. Derewenda, Z. S., and Sharp, A. M. (1993) Trends Biochem. Sci. 18, 20 –25 membrane, which must remain intact. 26. Blow, D. (1990) Nature 343, 694 – 695 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Degradation of Lipid Vesicles in the Yeast Vacuole Requires Function of Cvt17, a Putative Lipase

Download PDF
5 pages

Loading next page...
 
/lp/unpaywall/degradation-of-lipid-vesicles-in-the-yeast-vacuole-requires-function-00kHf5XutE

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.c000739200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 1, Issue of January 19, pp. 2083–2087, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Degradation of Lipid Vesicles in the Yeast Vacuole Requires Function of Cvt17, a Putative Lipase* Received for publication, October 18, 2000, and in revised form, November 7, 2000 Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.C000739200 Sarah A. Teter‡, Kimberly P. Eggerton‡, Sidney V. Scott§, John Kim‡, April M. Fischer‡, and Daniel J. Klionsky‡¶ From the ‡University of Michigan, Department of Biology, Ann Arbor, Michigan 48109 and the §Section of Microbiology, University of California, Davis, California 95616 The vacuole/lysosome serves an essential role in al- cytoplasm to the vacuolar lumen. Cvt and autophagy employ many of the same molecular components and are mechanisti- lowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are cally related (3– 6). Both pathways involve the formation of key proteins in this process. In Saccharyomces cerevi- double-membrane cytosolic vesicles, sequestering either pre- siae, some resident vacuolar hydrolases are delivered by cursor aminopeptidase I (prAPI) specifically, or in the case of the cytoplasm to vacuole targeting (Cvt) pathway, which autophagy, also enveloping bulk cytosol in a nonselective man- shares mechanistic features with autophagy. Autophagy ner. Fusion of these vesicles with the vacuole results in the is a degradative pathway that is used to degrade and release of single-membrane subvacuolar vesicles within the recycle cellular components under starvation condi- lumen. These pathways require a mechanism for specific lysis tions. Both the Cvt pathway and autophagy employ dou- of the internalized vesicles, so that vesicle cargo can be released ble-membrane cytosolic vesicles to deliver cargo to the into the vacuole lumen, and further require a mechanism for vacuole. As a result, these pathways share a common degradation of vesicle lipids. terminal step, the degradation of subvacuolar vesicles. To understand the molecular basis of these import and deg- We have identified a protein, Cvt17, which is essential radation pathways, we carried out a genetic screen for mutants for this membrane lytic event. Cvt17 is a membrane defective in delivery of prAPI to the vacuole. We isolated a glycoprotein that contains a motif conserved in ester- series of mutants, cvt, which accumulate the precursor form of ases and lipases. The active-site serine of this motif is API. The cvt mutants overlap with mutants defective in auto- required for subvacuolar vesicle lysis. This is the first phagy. The majority of these mutants are blocked at a stage characterization of a putative lipase implicated in vac- involving formation of the sequestering vesicle (reviewed in uolar function in yeast. Ref. 1). One mutant, cvt17, was found to be blocked in the breakdown of subvacuolar vesicles, suggesting that Cvt17 acts at a late stage of the import process (6). We report in this paper One fundamental role of the yeast vacuole is in the recycling the cloning of the gene encoding CVT17. Immunological and of biological macromolecules. The vacuole, like the lysosome in biochemical studies demonstrate that Cvt17 is a glycosylated, animal cells, is the primary site of degradation. Our under- integral membrane protein that transits through the secretory standing of the hydrolytic vacuolar enzymes that serve in pro- pathway. Cvt17 contains a domain that is conserved among tein turnover is well advanced. Progress has also been made in lipases of the a/b-hydrolase-fold superfamily. Mutation of the elucidating mechanisms that deliver the substrates of these putative lipase active site abolishes Cvt17 function in the Cvt vacuolar hydrolases. While research has focused on the biosyn- and autophagy pathways, suggesting that lipase activity is thesis and function of the vacuolar proteases, little is known critical to the role of this protein in these import processes. about how lipids are recycled in this organelle, and a lipase that functions in membrane recycling has not been identified. EXPERIMENTAL PROCEDURES Nearly all vacuolar/lysosomal delivery pathways involve Strains and Media—Wild type yeast strain SEY6210, was described packaging of cargo within membrane-enclosed transport com- previously (7), as were the mutant strains THY32 (cvt17-1; Ref. 3), partments. Because the vacuole/lysosome serves as the final MGY101 (apg5D::LEU2; Ref. 8), TVY1 (pep4D::LEU2; Ref. 9), WSY99 destination for these numerous vesicle-mediated transport (ypt7D::HIS3; Ref. 10), and NNY20 (apg1D::LEU2; Ref. 11). Strain WPHYD7 SEY6210 aut7D::LEU2 will be described elsewhere. The pathways, the issue of how membranes reaching the vacuole cvt17D strain is described below. Yeast cells were grown as described are recycled is an important one. Macroautophagy is the major previously (12). degradative process in eukaryotes and is essential during star- Materials—We followed standard procedures to prepare antiserum vation conditions (1). In yeast, autophagy overlaps with a bio- to Cvt17, using synthetic peptides corresponding to amino acids 131– synthetic process, the Cvt pathway, that delivers the hydrolase 152 and 295–312 (Multiple Peptide Systems, San Diego, CA). Addi- aminopeptidase I (API ; Ref. 2) from its site of synthesis in the tional antisera were described previously (13–15). The copper-inducible promoter-based plasmid pCu426 was from Dr. Dennis J. Thiele (Uni- * This work was supported by Public Health Service Grant GM53396 from the National Institutes of Health (to D. J. K.). The costs of publi- sor API; Cvt, cytoplasm to vacuole targeting; Endo H, endoglycosidase cation of this article were defrayed in part by the payment of page H; ER, endoplasmic reticulum; GFP, green fluorescent protein; PMSF, charges. This article must therefore be hereby marked “advertisement” phenylmethylsulfonyl fluoride; SD-N, synthetic minimal medium lack- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ing nitrogen; ORF, open reading frame; SGD, Saccharomyces Genome ¶ To whom correspondence should be addressed. Tel.: 734-615-6556; Database; PCR, polymerase chain reaction. Fax: 734-647-0884; E-mail: [email protected]. Kim, J., Huang, W.-P., and Klionsky, D. J. (2001) J. Cell Biol. 152, The abbreviations used are: API, aminopeptidase I; prAPI, precur- in press This paper is available on line at http://www.jbc.org 2083 This is an Open Access article under the CC BY license. 2084 Cvt17, a Putative Lipase Required for Cvt and Autophagy versity of Michigan; Ref. 16). Other reagents are identical to those described previously (12, 17). Cloning, Disruption, and Mutagenesis of CVT17—The CVT17 gene was cloned by transforming the cvt17-1 strain with a plasmid genomic library and screening for complementation of the starvation-sensitive phenotype on plates containing phloxine B (18). A secondary screen for prAPI accumulation was carried out as described previously (2). Sub- cloning of a complementing plasmid identified YCR068w as the com- plementing CVT17 ORF. The cvt17D strain (KTYD17) was made by inserting the LEU2 gene into the CVT17 ORF of strain SEY6210. At the time of this analysis, the Saccharomyces Genome Database (SGD) se- quence had shown YCR068w as a 1.289-kilobase coding sequence. We amplified this region and subcloned it into pCu426 using PCR (all primer sequences available upon request). This construct (pCuCVT17DC(426)) failed to complement the prAPI defect of cvt17D. The construct was sequenced, and comparison to the SGD sequence revealed a single cytosine insertion in the PCR amplified gene, at nucleotide 956 of the ORF. Sequencing of the original genomic plasmid revealed an identical sequence to the PCR product, suggesting an error in the SGD sequence. A recent revision of the chromosome III sequence has corrected this error. The rectified CVT17 sequence shifts the frame of the coding se- quence, increasing the length of the encoded protein. The longer, full- length coding sequence was amplified by PCR and subcloned into FIG.1. The cvt17D strain accumulates Cvt bodies within the pCu426 to generate pCuCVT17(426). For analysis of Cvt17 expression vacuole. A, precursor API is enclosed within a membrane compart- driven by the native promoter, CVT17 was amplified by PCR and cloned ment in the cvt17D strain. Osmotically lysed spheroplasts (T) were into pRS416 and pRS426, with inclusion of 0.5 kilobase before the start separated into low speed supernatant (S) and pellet (P) fractions by a codon and 0.432 kilobase after the stop codon. A PCR-based overlap- 5,000 3 g centrifugation. P fractions were resuspended in lysis buffer extension strategy was used in site-directed mutagenesis of CVT17. alone or treated with proteinase K 6 Triton X-100. B, the Cvt vesicle Serine 332 was converted to alanine by changing the T at nucleotide 994 marker protein GFPAut7 accumulates in subvacuolar vesicles in to G. cvt17D. pep4D and cvt17D cells harboring the pCuGFPAUT7(416) plas- Other Procedures—Cell fractionation, immunoprecipitation of radio- mid were grown to mid-log phase. GFPAut7 expression was induced labeled proteins, nitrogen starvation sensitivity, prAPI protease sensi- with 10 mM CuSO for 3 h. Vacuoles were labeled with FM 4-64. tivity, and reversal in SD-N were performed as described previously (8, 19). For endoglycosidase H (Endo H) treatment, cells were labeled for 10 a pelletable fraction in cvt17D, as it was in the apg5D and ypt7D min with no chase. Immunoprecipitated proteins were resuspended in mutant strains (Fig. 1A). In the apg5D strain, prAPI was sen- 0.1% SDS, 50 mM sodium citrate, pH 5.5, 1 mM PMSF. After boiling for 7 min, an equal volume of 0.1% SDS and 0.1 M b-mercaptoethanol was sitive to exogenous protease, even in the absence of detergent, added, then boiled for 7 min. Denatured proteins were incubated over- consistent with a block in vesicle formation (8). In contrast, night at 37° C in the presence of 50 mM sodium citrate, pH 5.5, 16 mM ypt7D and cvt17D cells contained prAPI that was not degraded b-mercaptoethanol, 0.03% SDS, 5 mM PMSF, and 2 units of Endo H. A to the mature form in the presence of exogenously added pro- second immunoprecipitation recovered the Cvt17 proteins. teinase K unless detergent was also added, indicating that Fluorescence Microscopy—Construction of the GFPAut7 fusion pro- prAPI was within a membrane enclosed compartment (Fig. 1A; tein and FM 4-64 labeling of cells are described elsewhere. Cells were examined using a Nikon Eclipse E-800 fluorescence microscope. Images Ref. 21). These data, coupled with the vesicle accumulation were captured by a Hamamatsu C4742–98 digital camera. phenotype of cvt17-1, suggest that prAPI was accumulating within subvacuolar Cvt vesicles in the cvt17D strain. RESULTS To assess whether the vesicles accumulating in cvt17D are Cvt17 Is Required for Lysis of Subvacuolar Cvt Bodies and indeed Cvt bodies, we examined whether a Cvt vesicle marker Autophagic Bodies—Previous analysis of cvt17-1 by electron accumulated within subvacuolar vesicles during vegetative microscopy suggested a defect in the breakdown of subvacu- growth conditions. Aut7 is a component that is required for Cvt olar, prAPI-containing vesicles, called Cvt bodies (3, 6). We vesicle formation and is itself enclosed within Cvt vesicles (22). isolated the CVT17 gene and disrupted the chromosomal locus We have recently demonstrated the utility of a GFPAut7 con- to generate a cvt17D strain (see “Experimental Procedures”). struct as a marker for following the formation and movement of The null phenotype was accumulation of prAPI (Fig. 1A), as Cvt vesicles in vivo. To visualize the vacuole membrane, we seen previously with the cvt17-1 mutant. used FM 4-64, a lipophilic red dye. In cvt17D, GFPAut7 is To determine the site of action of Cvt17, we analyzed the localized to Cvt bodies, seen as punctate structures accumulat- state of precursor API in cvt17D. Yeast cells were converted to ing within FM 4-64-labeled vacuoles (Fig. 1B, top panels). Some spheroplasts and subjected to osmotic lysis. Addition of exoge- GFPAut7 punctate staining was also observed outside the vac- nous protease allowed us to determine whether the accumu- uole. Mutants in the PEP4 gene, encoding vacuolar proteinase lated prAPI was blocked at a stage prior to Cvt vesicle forma- A, accumulate subvacuolar vesicles, including Cvt bodies by tion/completion (protease-sensitive) or at a point following electron microscopy (5). Consistent with these previous obser- enclosure but prior to fusion with the vacuole or vesicle break- vations, GFPAut7-labeled Cvt bodies also accumulated in the down (protease-protected). As controls, we examined prAPI vacuoles of a pep4D strain (Fig. 1B, bottom panels) but did not protease sensitivity in two strains that are defective in prAPI accumulate in the vacuoles of wild type yeast (data not shown). import. Apg5 is part of a novel protein conjugation complex The FM 4-64 vacuolar staining in cvt17D differed from that in that is required for both the Cvt and autophagy pathways (20). other strains, including pep4D; we observed a relative increase The apg5D strain is blocked in the completion of Cvt vesicles in the number of FM 4-64-labeled compartments per cell in the and accumulates membrane-associated, but protease-sensitive, cvt17D strain (Fig. 1B). While we do not know the molecular prAPI (8). Ypt7 is a rab GTPase that is required for fusion of basis for the change in vacuolar morphology, the microscopy Cvt vesicles and autophagosomes with the vacuole (1). The data provide direct evidence that cvt17D accumulates Cvt bod- ypt7D mutant accumulates prAPI in a protease-resistant state ies within the vacuole. within completed cytosolic vesicles. Precursor API was found in While many molecular components are shared between the Cvt17, a Putative Lipase Required for Cvt and Autophagy 2085 FIG.2. The cvt17D strain is defective in autophagy. A, log phase cultures grown in 1% yeast extract, 2% peptone, 2% glucose (YPD) were shifted to medium lacking nitrogen (SD-N). Viability was determined by plating aliquots on YPD plates. B, indicated strains were shifted to SD-N as in A, with aliquots taken at indicated times. Crude lysates were subjected to immunoblot analysis with anti-API antiserum. FIG.3. The nucleophilic serine of a putative lipase domain is Cvt and autophagy pathways, some cvt mutants are not defec- required for Cvt17 function. A, wild type yeast or strain cvt17D with the indicated CVT17-containing plasmids were grown to mid-log stage. tive in autophagy (3, 12). To test whether cvt17D has a defect in Western blots with anti-API antiserum are shown. Plasmids: library, autophagy, we examined whether the strain can survive complementing library plasmid; DC-term. 2m, truncated CVT17 gene growth in nitrogen-depleted media. Because transport to the with the last 87 residues deleted cloned in pCu426; 2m and CEN, vacuole by autophagy is the primary mode for degradation of full-length CVT17 gene in pRS426 and pRS416, respectively; S332A 2m and S332A CEN, Cvt17 with Ser to Ala mutation in the putative cytoplasmic constituents under starvation conditions, the proc- catalytic site cloned in pRS426 and pRS416, respectively. B, sequence ess is essential for viability during nutrient limitation. Wild alignments of Cvt17 and related fungal proteins (see “Results” for type cells can withstand nutrient deprivation for long periods of details). Gaps in the alignment (-), fully conserved residues (*), and time, while autophagy mutants such as apg1D or protease- residues with strong (:) and weaker (.) similarity are indicated. Putative catalytic triad residues (S-D-H) are indicated by outlined boxes; only deficient strains such as pep4D show reduced viability follow- one of the conserved Asp residues is expected to be active. The gray box ing a shift from growth in nutrient-rich medium to nitrogen- indicates the sequence that matches the consensus lipase pattern. deficient medium (Fig. 2A). Like apg1D and pep4D, the cvt17D ClustalX software was used to align sequences. strain was starvation-sensitive. Growing yeast cells transport prAPI to the vacuole via Cvt vesicles, while under nutrient deprivation, prAPI can be trans- kDa, rather than the 49.9 kDa predicted in the original data ported by autophagosomes (4, 5). If cvt17D is defective in lysis base. The full-length gene is able to complement the cvt17D of autophagic bodies, as its starvation sensitivity suggests, we strain, as shown by the accumulation of mAPI (Fig. 3A, 2m and should see a block in prAPI maturation not only in rich media CEN). Importantly, the frameshift occurred just 59 to a consen- but also under starvation conditions. Indeed, both the Cvt and sus site that is conserved in lipases and esterases (EC 3.1.1.3). autophagic routes of delivery are blocked, as indicated by anal- The presence of this motif was revealed by a BLOCK search ysis of API following a shift to SD-N medium (Fig. 2B). Auto- using the full-length, correct Cvt17 sequence. The sequence phagy mutants display differential blocks in API import under from amino acid 324 to 338 matches the consensus pattern of nutrient-rich versus starvation conditions. For example, mu- serine-active lipases (PROSITE reference PS00120; [LIV]-X- tants such as aut7D that do not have a complete defect in [LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]; Fig. 3B). A autophagy accumulate prAPI in nutrient-rich media but are BLAST search identified related proteins in several distinct TM able to mature prAPI under starvation conditions (Ref. 19; Fig. fungi, including Candida albicans (GenBank accession num- 2B). In contrast, strains that have more severe autophagy ber AL033391; 43% identity), as well as the fission yeast Schiz- TM defects, including apg1D, accumulate prAPI even after pro- osaccharomyces pombe (GenBank accession number Z99753; longed growth in SD-N. The cvt17D strain did not exhibit a 48% identity) and the tomato pathogen Cladosporium fulvum TM rescue of the prAPI processing defect when cells were shifted to (GenBank accession number Y14554; 38% identity (23)). SD-N, indicating that it is defective in autophagy as well as the Alignments of the partial sequences over an area of significant Cvt pathway (Fig. 2B). From these results we conclude that homology are shown in Fig. 3B. Cvt17 is most similar to the Cvt17 is involved in the lysis of autophagic bodies, as well as subfamily of fungal lipases within the larger a/b-hydrolase-fold Cvt bodies. superfamily (24, 25). Triglcyeride lipases are lipolytic enzymes Putative Serine Nucleophile in a Consensus Lipase Active that hydrolyze the ester bond of triglycerides. In addition to the Site Is Required for Cvt17 Function—Our original analysis of conserved serine within the active site consensus motif, a his- CVT17 revealed an SGD error in the coding region of this ORF tidine residue and an aspartic acid residue act in hydrolysis in (see “Experimental Procedures”). We discovered that the pre- a charge-relay system (26). We found two well conserved as- dicted coding sequence that was given in the data base at that partic acid residues in Cvt17 and other related fungal genes as time did not complement the prAPI accumulation phenotype in well as a conserved histidine (Fig. 3B). cvt17D (Fig. 3A, DC-term. 2 m). Sequencing revealed the pres- To determine whether the serine of the putative lipase do- ence of an extra cytosine base in the gene, at nucleotide 956 of main is required for the function of Cvt17, we mutated the the ORF. This insertion shifts the predicted reading frame, serine at position 332 to an alanine residue. Western blot resulting in a protein with a predicted molecular mass of 58.5 analysis of API in the cvt17D strain that contained the mutated 2086 Cvt17, a Putative Lipase Required for Cvt and Autophagy FIG.5. Cvt17 is a short-lived glycoprotein. A, cells were labeled for 10 min, followed by immunoprecipitation with antiserum to Cvt17. Immunoprecipitated proteins were mock treated or treated with Endo H. B, wild type cells were labeled for 10 min. Crude extracts from FIG.4. Cvt17-specific antiserum recognizes a 70-kDa mem- samples collected at the indicated chase times were immunoprecipi- brane protein. A, cells from cvt17D, wild type and cvt17D cells har- tated with Cvt17-specific antiserum. boring 2m wild type CVT17 and S332A mutant plasmids were radiola- beled for 5 min followed by a 10-min chase. Crude extracts were immunoprecipitated with antiserum to Cvt17. B, spheroplasts of wild vation that Cvt17 is glycosylated allows us to infer the mem- type cells were osmotically lysed, and the resultant lysate (T) was brane topology of the protein. All three putative glycosylation centrifuged at 13,000 3 g to yield supernatant (S13) and pellet (P13) sites are C-terminal to the transmembrane domain, suggesting fractions. S13 was further separated into high speed S100 and P100 that Cvt17 is a type II integral membrane protein. Upon inte- fractions by centrifugation at 100,000 3 g. Samples were subjected to immunoblot analysis with antisera to Cvt17, the vacuolar membrane- gration into the ER membrane, the N-terminal 13 amino acids associated protein Vac8, and the cytosolic protein phosphoglycerate would remain exposed to the cytosol, with the majority of the kinase (PGK). protein located in the lumen of the ER. The phenotype of the cvt17 mutant, accumulation of sub- gene (cvt17S332A) showed accumulation of the precursor form vacuolar vesicles, coupled with its characterization as a secre- of API, even when the mutant gene was expressed from a high tory pathway protein suggests that Cvt17 might function copy plasmid (Fig. 3A). In contrast, the wild type gene allowed within the vacuole lumen. Accordingly, we attempted to local- for prAPI maturation on both high and low copy plasmids. ize the Cvt17 protein. Subcellular fractionation based on veloc- Thus, Cvt17 cannot function to break down subvacuolar vesi- ity sedimentation indicated that Cvt17 was predominantly lo- cles and allow prAPI to be released into the vacuole without calized within a 13,000 3 g fraction (Fig. 4B). The P13 fraction this serine within the G-X-S-X-G lipase motif. contains various subcellular compartments, including the vac- Cvt17 Is a Glycosylated, Integral Membrane Protein—The uole. To directly determine whether Cvt17 was located within potential assignment of Cvt17 as a lipase required for the the vacuole, we purified vacuoles from yeast spheroplasts. degradation of subvacuolar vesicles led us to examine its bio- However, we were unable to detect Cvt17 in the vacuolar synthesis. To this end, we generated polyclonal antiserum fraction using this technique (data not shown). As an alterna- against the protein. Immunoprecipitation revealed a single tive approach, we fractionated cell lysates on an OptiPrep predominant band of ;70 kDa. This band was not detected in density gradient. Using this technique, in the presence of sub- the cvt17D strain, and expression of CVT17 from a high copy stantial amounts of protease inhibitors, we were able to detect plasmid greatly increased the abundance of this protein (Fig. small amounts of Cvt17 in fractions that contained the vacuo- 4A, 2m lanes), suggesting that the serum was specific for Cvt17. lar protein marker alkaline phosphatase (data not shown). We also immunoprecipitated Cvt17 from the strain harboring However, the majority of the Cvt17 was found in fractions that the mutant protein, which lacked the putative catalytic serine were distinct from the vacuole fractions. Thus, Cvt17 is found (cvt17S332A 2m). The altered protein was recovered in equiv- predominately in a membrane fraction residing outside the alent amounts to the wild type protein, indicating that the vacuole under steady-state conditions. single point mutation did not affect the stability of the protein. To examine the localization of Cvt17 in vivo, we engineered Analysis of the Cvt17 primary sequence indicates a stretch of GFP fusions with the protein at both the N and C termini. Both hydrophobic amino acids (residues 13–35) that might serve as constructs only partially complemented the Cvt trafficking de- a membrane anchor for the protein. Following osmotic lysis and fect of the cvt17D strain, as assessed by API Western blotting subcellular fractionation, Cvt17 was predominately localized to (data not shown). Furthermore, we were unable to visualize a 13,000 3 g pelletable fraction (Fig. 4B). We investigated strong fluorescent signals anywhere in cells containing these whether this localization was due to direct interaction with a GFP fusion constructs. An explanation for this lack of signal membrane by subjecting the P13 fraction to different wash might be an inherent instability of Cvt17. To examine stability conditions. Alkali extraction efficiently removed the peripheral of Cvt17 directly, we carried out a pulse-chase analysis. Yeast membrane protein Vma2 from the membrane but had no effect cells were labeled with [ S]methionine/cysteine and subjected on Cvt17 or the integral membrane protein Dpm1 (data not to a nonradioactive chase. At each time point, samples were shown). Treatment with the detergent Triton X-100 resulted in removed and immunoprecipitated. We found that Cvt17 was the solubilization of all three proteins. These results suggest unstable in wild type cells; the protein was degraded with a that Cvt17 is an integral membrane protein. half-life of ;45 min (Fig. 5B). The inherent instability of this The predicted molecular mass of Cvt17 is 58.5 kDa; however, protein made its localization problematic as much of it was the protein migrated with a molecular mass of 70 kDa following degraded during fractionation procedures. SDS-polyacrylamide gel electrophoresis (Fig. 4A). The Cvt17 DISCUSSION sequence has three predicted N-linked glycosylation sites. To determine whether the aberrant migration was due to glycosy- We have identified a protein that is required for the turnover lation, we treated immunoprecipitated Cvt17 with Endo H. of membrane vesicles in the vacuole of yeast. Sequence analysis Following treatment with the deglycosylating enzyme, the mo- of this protein reveals the presence of a domain found in lipases lecular mass of Cvt17 shifted to ;61 kDa (Fig. 5A). This change and esterases, and we have confirmed the importance of this in molecular mass correlates well with the predicted removal of domain by using site-directed mutagenesis of a putative active three N-linked carbohydrate side chains. This post-transla- site serine residue within the lipase motif. Phenotypic analysis tional modification reveals that Cvt17 must reside at least of yeast expressing the altered Cvt17 protein shows that the temporarily in the secretory pathway. Furthermore, the obser- protein is inactive in the Cvt pathway without this residue. The Cvt17, a Putative Lipase Required for Cvt and Autophagy 2087 cvt17D mutant is unable to degrade both Cvt bodies and auto- Cvt17 function, and specifically the function of a lipase active phagic bodies and is extremely sensitive to starvation (Fig. 2A). site domain, is required for the lysis of subvacuolar Cvt and The ability to degrade membranes that are released into the autophagic bodies. Characterization of this protein is an im- vacuole lumen is required for starvation survival. In the ab- portant first step in understanding vacuolar function in the sence of this degradative capacity, the cell is unable to break turnover of lipids and in the terminal steps of the Cvt and down lipid and, as a result, is unable to access cargo contained autophagic pathways. Continued analysis of Cvt17 will provide within autophagic bodies. The occurrence of genes with simi- important information about membrane recycling in the larity to CVT17 in other fungi suggests that this may be a vacuole. conserved protein in the kingdom. Acknowledgments—We thank Jacob Teter for technical assistance, The maintenance of membrane-bound vesicles in the cvt17D Bob Fuller for use of the fluorescence microscope, and E. J. Brace for strain suggests a role for the protein in degrading a phospho- help with microscopy. We thank Maria Hutchins for comments on the lipid bilayer. Sequence comparisons, however, place the protein manuscript and for technical assistance. in a class of hydrolytic enzymes that act primarily on triglyc- REFERENCES erides, not phospholipids. It will be necessary to perform bio- 1. Kim, J., and Klionsky, D. J. (2000) Annu. Rev. Biochem. 69, 303–342 chemical studies with the purified enzyme to investigate its 2. Harding, T. M., Morano, K. A., Scott, S. V., and Klionsky, D. J. (1995) J. Cell substrate specificity. To function properly, lipases must be Biol. 131, 591– 602 targeted to the appropriate substrates. The membrane source 3. Harding, T. M., Hefner-Gravink, A., Thumm, M., and Klionsky, D. J. (1996) J. Biol. Chem. 271, 17621–17624 of Cvt vesicles and autophagosomes is not known. If Cvt17 is 4. Scott, S. V., Hefner-Gravink, A., Morano, K. A., Noda, T., Ohsumi, Y., and incorporated into Cvt vesicles and autophagosomes, its topol- Klionsky, D. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12304 –12308 5. Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., and Ohsumi, Y. (1997) J. Cell ogy would place the Cvt17 active site on the outer surface of the Biol. 139, 1687–1695 Cvt and autophagic bodies. To prevent membrane degradation 6. Scott, S. V., Baba, M., Ohsumi, Y., and Klionsky, D. J. (1997) J. Cell Biol. 138, during transit, it is possible that Cvt17 becomes active only in 37– 44 7. Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. the vacuole. If this is the case, the lytic activity of this protein Biol. 8, 4936 – 4948 must be suppressed during transit. Some aspect of the vacuolar 8. George, M. D., Baba, M., Scott, S. V., Mizushima, N., Garrison, B. S., Ohsumi, Y., and Klionsky, D. J. (2000) Mol. Biol. Cell 11, 969 –982 milieu may then activate Cvt17 as is thought to be the case for 9. Gerhardt, B., Kordas, T. J., Thompson, C. M., Patel, P., and Vida, T. (1998) proteinase A. If Cvt17 acts directly to degrade subvacuolar J. Biol. Chem. 19, 15818 –15829 vesicles, it should reside within the vacuole. Subcellular frac- 10. Wurmser, A., and Emr, S. (1998) EMBO J. 17, 4930 – 4942 11. Matsuura, A., Tsukada, M., Wada, Y., and Ohsumi, Y. (1997) Gene (Amst.) 192, tionation did reveal a small population of vacuolar Cvt17 that 245–250 may play a primary role in directly degrading Cvt and auto- 12. Scott, S. V., Nice, D. C., III, Nau, J. J., Weisman, L. S., Kamada, Y., Keizer- Gunnink, I., Funakoshi, T., Veenhuis, M., Ohsumi, Y., and Klionsky, D. J. phagic bodies. Most of the protein, however, did not cofraction- (2000) J. Biol. Chem. 275, 25840 –25849 ate with this organelle. In agreement with these results, the 13. Klionsky, D. J., Cueva, R., and Yaver, D. S. (1992) J. Cell Biol. 119, 287–299 rapid Cvt17 degradation that we observed (Fig. 5B) was inde- 14. Tomashek, J. J., Sonnenburg, J. L., Artimovich, J. M., and Klionsky, D. J. (1996) J. Biol. Chem. 271, 10397–10404 pendent of the major vacuole proteases (data not shown). 15. Wang, Y. X., Catlett, N. L., and Weisman, L. S. (1998) J. Cell Biol. 140, The reasons for maintaining a sizable population of Cvt17 1063–1074 16. Labbe ´ , S., and Thiele, D. J. (1997) Methods Enzymol. 306, 145–153 outside the vacuole are as yet obscure. It is possible that Cvt17 17. Noda, T., Kim, J., Huang, W.-P., Baba, M., Tokunaga, C., Ohsumi, Y., and performs some additional unknown function outside the vacu- Klionsky, D. J. (2000) J. Cell Biol. 148, 465– 479 ole. Another possibility is that Cvt17 acts exclusively outside 18. Tsukada, M., and Ohsumi, Y. (1993) FEBS Lett. 333, 169 –174 19. Abeliovich, H., Dunn, W. A., Jr., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. the vacuole and only indirectly influences the lytic competence 151, 1025–1033 of subvacuolar vesicles; Cvt17 might act as a lipase to specifi- 20. Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., and Ohsumi, Y. (1998) Nature 395, 395–398 cally alter the lipid composition of Cvt vesicles and autophago- 21. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., and Klionsky, D. J. (1999) somes as they form. Specific lipid composition might be a re- Mol. Biol. Cell 10, 1337–1351 quirement for the subsequent lysis of Cvt bodies and 22. Huang, W.-P., Scott, S. V., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. 275, 5845–5851 autophagic bodies. Lipids could even provide the molecular 23. Coleman, M., Henricot, B., Arnau, J., and Oliver, R. P. (1997) Mol. Plant basis for recognition within the vacuole lumen, allowing an Microbe Interact. 10, 1106 –1109 24. Cousin, X., Hotelier, T., Giles, K., Toutant, J. P., and Chatonnet, A. (1998) alternate vacuolar lipase to distinguish between subvacuolar Nucleic Acids Res. 26, 226 –228 vesicles destined for degradation and the bounding vacuolar 25. Derewenda, Z. S., and Sharp, A. M. (1993) Trends Biochem. Sci. 18, 20 –25 membrane, which must remain intact. 26. Blow, D. (1990) Nature 343, 694 – 695

Journal

Journal of Biological Chemistry – Unpaywall

Published: Jan 1, 2001

There are no references for this article.