Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/unpaywall/the-ccz1-mon1-protein-complex-is-required-for-the-late-step-of-CfPI1kNCeH
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.m208191200
- Publisher site
- See Article on Publisher Site
Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 49, Issue of December 6, pp. 47917–47927, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways* Received for publication, August 9, 2002, and in revised form, September 30, 2002 Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M208191200 Chao-Wen Wang‡, Per E. Stromhaug‡, Jun Shima‡§, and Daniel J. Klionsky From the ‡Department of Molecular, Cellular, and Developmental Biology and the Department of Biological Chemistry and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109 resident hydrolases and their substrates arrive at this destina- Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of tion through a variety of sorting pathways. The main routes by aminopeptidase I (Ape1) via the cytoplasm to vacuole which vacuolar hydrolases are delivered to this organelle are targeting (Cvt) pathway. The mon1 and ccz1 strains the carboxypeptidase Y (CPY), alkaline phosphatase (ALP), also displayed defects in autophagy and pexophagy, and multivesicular body (MVB) pathways, which involve tran- degradative pathways that share protein machinery sit through a portion of the secretory pathway, and the cyto- and mechanistic features with the biosynthetic Cvt plasm to vacuole targeting (Cvt) pathway by which the cargo pathway. Further analyses indicated that Mon1, like molecules are packaged as cytosolic membrane-bound interme- Ccz1, was required in nearly all membrane-trafficking diates (2, 3). Resident proteins are also transmitted by inher- pathways where the vacuole represented the terminal itance from mother cell vacuoles to daughter cells during cell acceptor compartment. Accordingly, both deletion division (4). Substrates enter the vacuole through endocytosis, strains had kinetic defects in the biosynthetic delivery autophagy and the vacuole import and degradation pathway of resident vacuolar hydrolases through the CPY, ALP, (reviewed in Ref. 5). One common feature in all of these pro- and MVB pathways. Biochemical and microscopy stud- cesses is membrane fusion. The membrane fusion mechanism ies suggested that Mon1 and Ccz1 functioned after acts to ensure specificity for the directed movement of proteins transport vesicle formation but before (or at) the fusion while also maintaining the distinct composition of each or- step with the vacuole. Thus, ccz1 and mon1 are the ganelle within the highly compartmentalized eukaryotic cell. first mutants identified in screens for the Cvt and Apg The cytoplasm to vacuole targeting pathway that is used to pathways that accumulate precursor Ape1 within com- deliver the soluble hydrolase aminopeptidase I (Ape1) to the pleted cytosolic vesicles. Subcellular fractionation and vacuole has been under investigation (for reviews see Refs. 2, 5, co-immunoprecipitation experiments confirm that Mon1 and 6). Under vegetative conditions, precursor Ape1 (prApe1) and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and is assembled into a large Cvt complex composed in part of Mon1 tagged with a fluorescent marker indicated that the multiple prApe1 dodecamers in the cytosol that becomes en- Ccz1-Mon1 complex peripherally associated with a peri- wrapped within a double-membrane Cvt vesicle (7). Upon com- vacuolar compartment and may attach to the vacuole pletion, the cytosolic Cvt vesicle targets to the vacuole. The membrane in agreement with their proposed function in outer membrane of the Cvt vesicle fuses with the vacuole fusion. membrane and the intact inner vesicle (Cvt body) passes into the vacuole lumen (8). The Cvt body is ultimately broken down by resident vacuolar hydrolases, resulting in the release and Compartmentalization allows eukaryotic cells to regulate maturation of prApe1. Precursor Ape1 is transported to the intracellular functions by separating competing reactions and vacuole by another pathway, termed autophagy (Apg), under localizing enzymes and substrates at specific locations within starvation conditions (2, 9). In the Apg pathway, portions of the cell. Efficient compartmentalization necessitates dynamic cytoplasm are sequestered within relatively larger double protein trafficking processes by which cells are able to establish membrane vesicles (autophagosomes) that are also targeted to and maintain the identity and function of each organelle. The the vacuole (7). Although Apg is a degradative process, mu- vacuole (lysosome) of the yeast Saccharomyces cerevisiae plays tants defective in autophagy, apg/aut, overlap with cvt mutants a central role in the turnover of cytoplasmic organelles, degra- (10). Morphological and biochemical analyses further indicate dation of intracellular/extracellular components, and mainte- that the Cvt and Apg pathways use analogous mechanisms nance of cellular physiology (1). To carry out these functions, (2, 5, 9). the vacuole maintains a variety of degradative enzymes. Both To gain additional insight into the Cvt/Apg pathways, we screened a gene deletion library for mutants that are defective in prApe1 maturation. We found two mutants that are required * This work was supported by National Institutes of Health Public for Cvt/Apg import that had not been previously implicated in Health Service Grant GM53396 (to D. J. K.), the Lewis E. and Elaine Prince Wehmeyer Trust (to C.-W. W.), and a research fellowship from these pathways. The product of one of these genes, Ccz1, has the Science and Technology Agency of Japan (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” The abbreviations used are: CPY, carboxypeptidase Y; ALP, alka- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. line phosphatase; Ape1, aminopeptidase I; CFP, cyan fluorescent pro- § Present address: Yeast Laboratory, National Food Research Insti- tein; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent pro- tute, Tsukuba, Ibaraki 305-8642, Japan. tein; prApe1, precursor aminopeptidase I; PVC, pre-vacuolar ¶ To whom correspondence should be addressed: University of compartment; SMD, synthetic minimal medium with dextrose; SD/-N, Michigan, Dept. of Molecular, Cellular and Developmental Biology, Ann synthetic minimal medium with dextrose but lacking nitrogen; YFP, Arbor, MI 48109-1048. Tel.: 734-615-6556; Fax: 734-647-0884; E-mail: yellow fluorescent protein; ORF, open reading frame; MES, 4-morpho- [email protected]. lineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. This paper is available on line at http://www.jbc.org 47917 This is an Open Access article under the CC BY license. 47918 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex TABLE I Yeast strains used in this study Strain Genotype Reference SEY6210 MAT leu2–3,112 ura3–52 his3–200 trp1–901 lys2–801 suc2–9 GAL (48) CWY3 SEY6210 ccz1::HIS5 This study JSY1 SEY6210 mon1::HIS5 This study BY4742 MAT his3 leu2 lys2 ura3 ResGen ccz1 BY4742 ccz1::KanMX ResGen mon1 BY4742 mon1::KanMX ResGen D3Y102 SEY6210 vac8 (31) NNY20 MATa ura3 trp1 leu2 apg1::LEU2 (31) vps5 SEY6210 vps5 (48) CWY40 SEY6210 vam3::TRP1 This study WSY99 SEY6210 ypt7::HIS3 (49) VDY101 SEY6210 apg7::LEU2 (37) PSY35 SEY6210 MON1-HA::TRP1 This study PSY36 SEY6210 CCZ1-HA::TRP1 This study PSY42 SEY6210 CCZ1-YFP::HIS3 This study PSY44 SEY6210 CCZ1-CFP::KanMX This study PSY45 SEY6210 CCZ1-CFP::KanMX pCUP1-YFP-MON1::URA3 This study PSY46 SEY6210 CCZ1-GFP::HIS3 This study PSY47 SEY6210 MON1-GFP::HIS3 This study TVY1 SEY6210 pep4::LEU2 (50) against Ape1 (15), Prc1 (16), and Pep4 (16) have been described. Anti- been suggested to be involved in multiple trafficking pathways sera against Pgk1, Ypt7, and Anp1 were provided by Dr. Jeremy Thor- to the vacuole (11). Overexpression of the Rab protein Ypt7 ner (University of California, Berkeley, CA), Dr. William Wickner rescues the sensitivity to calcium, caffeine, and zinc observed (Dartmouth Medical School, Hanover, NH), and Dr. Sean Munro (MRC K127E with the ccz1 strain. The Ypt7 mutant has been identi- Laboratory of Molecular Biology, Cambridge, UK), respectively. Anti- fied as a specific mutation that suppresses the ccz1 phenotype bodies against Pho8, Dpm1, and Pep12 were obtained from Molecular (12). Co-immunoprecipitation data further support the physical Probes, and the anti-HA antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). To prepare antiserum against interaction between Ccz1 and Ypt7 (12). The mon1 strain is Mon1, the NH terminus of the Mon1 ORF (1–585 bp) was PCR- sensitive to monensin and brefeldin A (13), but is otherwise 2 amplified and fused to the COOH terminus of the maltose-binding uncharacterized. In this study, we show that strains lacking protein. The resulting plasmid was transformed into E. coli strain either of these two proteins have similar phenotypes. Both BL21. Fusion protein purification and antiserum generation were as Mon1 and Ccz1 are required not only for the Cvt/Apg pathways described (17). but also other vacuole biogenesis processes including the sort- Screening the Haploid Gene Deletion Library—A MAT haploid gene ing of newly synthesized vacuolar proteins through the CPY, deletion library was obtained from ResGen/Invitrogen Corporation (Huntsville, AL). The mutants provided from the company were inocu- ALP, and MVB pathways and endocytosis. Biochemical and lated on YPD plates and incubated at 30 °C for 12–24 h. The cells on morphological evidence further indicate that the Cvt/Apg path- YPD plates were collect and resuspended in 50 l of MURB (50 mM ways are blocked at a stage after the formation of the seques- NaPO ,25mM MES, pH 7.0, 1% SDS, 3 M urea, 0.5% -mercaptoetha- tering vesicles but prior to their fusion with the vacuole. These nol, 1 mM NaN , and 0.05% bromphenol blue) and converted into crude studies also suggest that Ccz1 and Mon1 co-localize to a unique cell extracts by glass bead lysis and boiling. The extracts were subjected membrane and that they physically interact. Finally, we dem- to immunoblot analysis using anti-Ape1 antisera. onstrate the in vivo localization of these two proteins to a Disruption, Epitope Tagging, and Gene Cloning—The chromosomal perivacuolar compartment and the vacuole membrane, a site MON1 and CCZ1 loci were deleted by a PCR-based, one-step procedure (18). In brief, the corresponding auxotrophic marker was amplified from consistent with their proposed role in fusion. the pME3 or pFA6a knockout plasmids by PCR using oligonucleotides that contained sequences outside of the marker, flanked by sequences EXPERIMENTAL PROCEDURES that encode regions at the beginning and end of the corresponding Strains, Media, and Growth Conditions—The yeast strains used in ORFs. PCR products were used to transform yeast strain SEY6210. this study are listed in Table I. Synthetic minimal medium (SMD) Putative knockout strains were checked by Western blot for the Ape1 contained 0.67% yeast nitrogen base without amino acids, 2% glucose, phenotype. Similar strategies were applied for the chromosomal HA and auxotrophic amino acids and vitamins as needed. Nitrogen starva- and fluorescent protein tagging. To clone the MON1 and CCZ1 genes, tion medium (SD-N) contained 0.17% yeast nitrogen base without both ORFs and their upstream/downstream sequences were PCR-am- amino acids and ammonium sulfate and 2% glucose. YPD medium plified using genomic DNA as template. The resulting PCR products for contained 1% yeast extract, 2% peptone, and 2% glucose. S. cerevisiae MON1 include 360 bp before the sequence encoding the start codon and strains were grown at 30 °C. Yeast cells used for this study were grown 405 bp after the stop codon. The fragments were digested with SacI and in the appropriate SMD medium to mid-log (OD of 0.6). SmaI and inserted into the SacI and SmaI site of the pRS416/426 vector Reagents and Antisera/Antibodies—Reagents for growth medium to generate plasmids pMON1(416/426). The PCR products for the clon- were from Difco Laboratories (Detroit, MI). DNA restriction enzymes, ing of CCZ1 contain 300-bp upstream and 700-bp downstream of the T4 DNA ligase and calf intestinal alkaline phosphatase were obtained CCZ1 ORF. The PCR products were digested with KpnI to generate from New England Biolabs, Inc. (Beverly, MA). Tran[ S] label was pCCZ1(416/426). To construct COOH-terminal HA epitope-tagged Ccz1, obtained from ICN (Costa Mesa, CA). Oxalyticase was from Enzogenet- TM the CCZ1 ORF was PCR-amplified using pCCZ1(416) as a template. ics (Corvallis, OR). OptiPrep was from Accurate Chemical and Sci- TM The resulting PCR product was digested and inserted into pRS416HA entific Corp. (Westbury, NY). Complete EDTA-free protease inhibitor and pRS426HA that contains a 3HA epitope (19). To construct an was obtained from Roche Molecular Biochemicals. The pME3 vector NH -terminal YFP fusion to Mon1, the MON1 ORF was PCR-amplified containing the Schizosaccharomyces pombe HIS5 auxotrophic marker using pMON1 (416) as a template. The resulting PCR products were was a gift from Dr. Neta Dean (State University of New York, Stony inserted into pCuYFP (306) to generate pCuYFP-MON1 (306). The Brook, NY). The pFA6a knockout and tagging vectors containing TRP1, HIS3,or KanMX markers were generous gifts from Dr. Mark Longtine construct was linearized with KpnI and transformed into strain PSY44 (Oklahoma State University) (14). The CFP (pDH3) and YFP (pDH5) to replace endogenous MON1 with pCuYFP-MON1 (strain PSY45). The plasmids were from the Yeast Resource Center (University of plasmids pCvt19-CFP(414) (20), pSte3-GFP(316) (21), pCuGFP-Aut7 Washington). FM 4-64 dye was obtained from Molecular Probes (416) (22), pGFP-Pho8(426) (23), and pSna3-GFP(416) (24) were de- (Eugene, OR). All other reagents were from Sigma-Aldrich. Antisera scribed previously. All oligonucleotide sequences and additional details Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex 47919 of the plasmid constructions will be provided upon request. for12hat4 °C in a Sorvall Th-641 rotor. Samples were collected from Immunoblot Analysis, Pulse/Chase Labeling, and Immunoprecipita- the top of the gradients into 14 fractions. The fractions were trichloro- tion—Immunoblot analysis was carried out essentially as described acetic acid-precipitated and washed twice with acetone followed by previously (25). For kinetic analysis of Prc1, yeast cells were grown to immunoblot analyses. an OD of 1.0 and converted into spheroplasts. The spheroplasts from Native Immunoprecipitation—The protocol for co-immunoprecipita- 20 OD units of cells were resuspended in 300 l of SMD medium tion with Ccz1-HA was modified from a previously described procedure containing 1.3 M sorbitol, and labeled with 20 Ci of Tran[ S] label for (12). In brief, 10 OD units of log-phase cells were lysed with glass 5 min, followed by a chase reaction in SMD containing 1.3 M sorbitol, beads in lysis buffer (50 mM HEPES, pH 7.4, 150 mM KCl, 1 mM EDTA, 0.2% yeast extract, 4 mM methionine, and 2 mM cysteine at a final 0.5% Triton X-100) with the addition of protease inhibitor mixture and density of 2.0 OD /ml. Samples were removed at the indicated time 1mM phenylmethylsulfonyl fluoride. After a 10-min solubilization on points and 1 mM NaN was added to stop the reaction. The samples ice, total cell lysates were centrifuged at 13,000 g for 15 min at 4 °C. were subjected to a 5,000 g centrifugation for 3 min. The resulting To the resulting supernatant, 10 l of anti-HA antiserum was added supernatant and pellet fractions were separately precipitated with 10% followed by incubation with protein A-Sepharose at 4 °C overnight. trichloroacetic acid. Trichloroacetic acid precipitates were resuspended Sepharose beads were washed with lysis buffer a total of eight times. in MURB buffer and subjected to immunoprecipitation as described Bound proteins were eluted in MURB followed by SDS-PAGE and previously (25). For kinetic analyses of Ape1, Pep4, and Ste3, yeast cells Western blot analysis. were grown to an OD of 1.0 in SMD medium. Cells (20 OD units) Microscopy—All strains used for microscopy were grown in SMD 600 600 were resuspended in 300 l of SMD medium and labeled with 20 Ci of medium to mid-log phase. In vivo FM 4 – 64 staining was performed as Tran[ S] label for 5–10 min, followed by a chase reaction as above at a described previously (27). Microscopy analysis was performed using a final density of 20 OD /ml. Samples were removed at the indicated Nikon E-800 fluorescent microscope (Mager Scientific Inc., Dexter, time points and precipitated with 10% trichloroacetic acid. Crude ex- MI). Images were captured by an ORCA II CCD camera (Hamamatsu tracts were prepared by glass bead lysis and subjected to immunopre- Corp., Bridgewater, NJ) using Openlab 3 software (Improvision, Inc., cipitation as described previously (25). Lexington, MA). Analyses of the Cvt Pathway and Autophagy—Cell viability and starvation curves and peroxisome degradation rates were determined RESULTS as described previously (17). The membrane flotation assay was per- Mon1 and Ccz1 Are Required for the Cvt, Autophagy, and formed essentially by the method described previously (26) with minor Pexophagy Pathways—Although various cvt, apg, and aut mu- modifications. Spheroplasts derived from the mon1 strain were resus- pended in PS200 lysis buffer (20 mM PIPES, pH 6.8, 200 mM sorbitol) tants defective in the Cvt and Apg pathways have been isolated containing 5 mM MgCl at a spheroplast density of 20 OD /ml. The and analyzed (reviewed in Refs. 5 and 28), many questions 2 600 lysate was centrifuged at 13,000 g for 5 min at 4 °C. The pellet concerning these pathways remain to be answered. We are fractions from 10 OD units of cells were resuspended in 100 lof15% interested in the molecular mechanism governing the dynamic Ficoll-400 (w/v) in lysis buffer with or without the addition of 0.2% aspects of the Cvt and Apg pathways. We reasoned that the Triton X-100. The resuspended pellet fractions were overlaid with 1 ml identification of additional mutants would provide further in- of 13% Ficoll-400 in lysis buffer and then overlaid with 200 lof2% Ficoll-400 in lysis buffer. The resulting step gradient was subjected to sight into the protein machinery of these processes. Accord- centrifugation at 13,000 g for 10 min at 4 °C. The top 500 l was ingly, we screened a haploid gene deletion library based on the designated as the float fraction (F), the remaining solution was consid- accumulation of prApe1, a cargo protein that is delivered to the ered as the nonfloat fraction (NF), and the gradient pellet was desig- vacuole through the Cvt/Apg pathways. Among the new mu- nated as the pellet fraction (P). The three fractions were trichloroacetic tants identified, mon1 and ccz1 showed a complete block in acid-precipitated, washed twice with acetone, and analyzed by immu- prApe1 maturation. Although mon1 has not been previously noblot. The protease protection assay was performed as described pre- viously (17). In brief, log-phase cultures were subjected to osmotic lysis reported as having a role in the Cvt pathway, complementation in PS200 containing 5 mM MgCl . The lysates were centrifuged at analyses indicate that CCZ1 is allelic with CVT16, a previously 13,000 g for 10 min, and the pellet fractions (P13) were resuspended uncharacterized CVT gene (10). The ccz1 mutant was origi- in lysis buffer in the presence or absence of 50 g/ml proteinase K nally identified due to its sensitivity to caffeine, calcium, and and/or 0.2% Triton X-100. Reactions were carried out on ice for 30 min zinc (29). It has also been shown that the strain displays a followed by trichloroacetic acid precipitation and immunoblot analysis. TM severe vacuole protein-sorting defect. Immunofluorescent data Subcellular Fractionation and OptiPrep Density Gradient Analy- sis—Mon1-HA cells expressing pCcz1-HA(416) were grown to mid-log suggest that Ccz1 localizes to the endosomal compartment, and phase (OD 0.6) in SMD medium. The cells were converted into it has been suggested to act in concert with the Rab protein spheroplasts and resuspended in PS200 lysis buffer containing 5 mM Ypt7 (11, 12). There has not been a published report describing TM MgCl and the Complete EDTA-free protease inhibitor mixture at a Mon1 function. The MON1 gene, YGL124c, encodes a 644- density of 20 OD /ml. After a preclearing spin (500 g, for 5 min, at amino acid protein with a predicted molecular mass of 73.5 4 °C), the total lysate was subjected to low-speed centrifugation kDa. A data base search indicates that Mon1 does not have (13,000 g for 10 min), resulting in the supernatant (S13) and pellet (P13) fractions. The S13 fraction was subjected to high speed centrifu- homology with other proteins of S. cerevisiae. However, possi- gation (100,000 g for 30 min at 4 °C) to generate the supernatant ble homologues having 24 –37% identity with Mon1 exist in (S100) and pellet (P100) fractions. The resulting fractions were sub- S. pombe, Caenorhabditis elegans, and Drosophila melanogaster. jected to immunoblot analysis. To examine membrane binding of Ccz1 has no significant homologues. Ccz1-HA and Mon1-HA the membrane fractions from lysed sphero- When wild type cells are grown under nutrient-rich condi- plasts were treated with 1 M KCl, 0.1 M Na CO (pH 10.5), 3 M urea, or 2 3 tions, the majority of Ape1 is present as the 50-kDa mature 1% Triton X-100 as described previously (17). OptiPrep™ density gra- dient analysis was performed using a modification of a previously form (Fig. 1A), although a small fraction is present as the described procedure (17). In brief, a Mon1-HA strain expressing 61-kDa precursor. In contrast, both the mon1 and ccz1 Ccz1-HA was grown to mid-log phase (OD 0.6) and converted into strains accumulated only the precursor form of Ape1. The spheroplasts. The spheroplasts were subjected to osmotic lysis in PS200 defect in prApe1 processing in these mutants was rescued by containing 1 mM EDTA, 1 mM MgCl , and a protease inhibitor mixture TM expressing either single or multicopy versions of the corre- (Complete EDTA-free protease inhibitor tablets, 1 g/ml leupeptin sponding genes on plasmids, confirming the essential roles of and 1 g/ml pepstatin A). The lysate was subjected to very low speed centrifugation (800 g for 5 min) to remove the remaining intact Mon1 and Ccz1 for the Cvt pathway (Fig. 1A). Precursor Ape1 spheroplasts. After this preclearing step, the crude lysate from 35 is delivered to the vacuole through autophagy under starvation OD units of cells was centrifuged at 100,000 g for 20 min at 4 °C. conditions. We utilized a starvation-sensitivity analysis to de- The resulting total membrane fraction was resuspended in 200 lof termine whether the mon1 and ccz1 strains were able to lysis buffer and then applied on top of a density gradient (12 ml, linear) carry out autophagy. Wild type cells, or mutants specific to the consisting of 10 –55% OptiPrep™ in PS200 lysis buffer containing 1 mM Cvt pathway, are starvation-resistant while mutants defective EDTA, 1 mM MgCl ,1mM dithiothreitol, and a protease inhibitor mixture. The gradients were subjected to centrifugation at 100,000 g for autophagy lose viability in the absence of nitrogen (17). As 47920 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex FIG.1. The ccz1 and mon1 strains are defective in the Cvt, autophagy, and pexophagy pathways. A, cloning and characterization of CCZ1 and MON1. Wild type (WT, SEY6210), ccz1 (CWY3), and mon1 (JSY1) strains and the knock- out strains expressing the respective single copy (CEN) or multicopy (2) plasmids were grown in SMD medium and analyzed by immunoblot against Ape1. B, mon1 and ccz1 strain are sensitive to nitrogen- starvation conditions. The wild type, apg1, and mon1 strains and the mon1 strain harboring pMON1(416) or the wild type and ccz1 strains were grown to mid- log phase in SMD medium and shifted to SD-N medium. At the indicated time, ali- quots were removed and spread onto YPD plates in triplicate. The number of viable colonies was counted after 2 days incuba- tion at 30 °C. C, mon1 and ccz1 mutants do not bypass the prApe1 accumulation de- fect when autophagy is induced. The vac8 (D3Y102), apg1 (NNY20), ccz1, and mon1 strains were grown to mid-log phase in SMD and shifted to SD-N me- dium. At the indicated time, aliquots were removed and subjected to immunoblot against Ape1. D, mon1 and ccz1 strains are defective for pexophagy. The wild type, ccz1 and mon1 strains in the BY4742 background were grown in YPD to mid-log phase, transferred to oleic acid medium to induce peroxisome production and shifted to SD-N. Aliquots were removed at the in- dicated times and analyzed by Western blot with antiserum to Fox3. shown in Fig. 1B, the wild type strain was resistant to starva- tion pathway, pexophagy, uses similar molecular components tion over the time course examined. In contrast, mon1 and as the Cvt and autophagy pathways (33). To investigate if ccz1 strains, similar to the apg1 mutant, displayed a rapid Mon1 and Ccz1 are also required for pexophagy, we induced the loss of viability in SD-N medium. Viability in the mon1 strain expression of peroxisomes by growing cells in oleic acid in the was restored when these cells expressed Mon1 from a CEN- wild type, mon1, and ccz1 strains, and then monitored the based plasmid. degradation of Fox3 after cells were shifted to glucose. Crude Starvation-sensitivity indicates that autophagy is not fully cell extracts were collected at the times indicated and exam- functional in the mon1 and ccz1 strains. Recently, however, ined by Western blot. In wild type cells, Fox3 levels decreased we have demonstrated that some mutants that are autophagy in SD-N, reflecting peroxisome degradation (Fig. 1D). In con- defective by this criterion are still able to induce the formation trast, both the mon1 and ccz1 strains maintained Fox3 at of autophagosomes under starvation conditions. For example, the initial level indicating a defect in peroxisome degradation. Therefore, we conclude that both Mon1 and Ccz1 are part of the the aut7 strain is starvation-sensitive but is able to induce the formation of small, abnormal autophagosomes in SD-N (30). In mechanism shared by the Cvt, autophagy, and pexophagy pathways. addition, some components of the Cvt and Apg pathways are only essential for one of these two pathways. For example, Vac8 Ccz1 and Mon1 Are Required for Multiple Vacuole Delivery Pathways—It has been reported that the ccz1 strain exhibits and Cvt9 are only required for the Cvt pathway whereas Apg17 appears to function only in autophagy (26, 31, 32). Accordingly, a severe vacuolar hydrolase sorting defect as well as a frag- mented vacuole phenotype (11). To gain a better understanding these types of mutants are able to mature prApe1 under star- vation conditions. We extended our analysis of autophagy by of vacuole protein delivery in the mon1 strain, we examined different cargo proteins that are targeted to the vacuole by examining the role of Ccz1 and Mon1 in prApe1 import under nutrient-deprivation conditions. Strains were grown in SMD to various mechanisms. Carboxypeptidase Y, Prc1, is transported to the vacuole through the CPY pathway, a transport itinerary mid-log phase, shifted to medium lacking nitrogen (SD-N), and the time course of prApe1 processing was examined by Western that includes the ER, Golgi complex, and endosome. In the wild type strain, Prc1 is matured (mPrc1) with a half-time of 5–10 blot (Fig. 1C). As expected, the vac8 strain showed a reversal of the prApe1 accumulation defect after cells were shifted to min. Approximately 5% of Prc1 is secreted from the cell under standard conditions used for this type of analysis (Fig. 2A). In SD-N. In contrast, the apg1 mutant that is defective for both the Cvt and Apg pathways was unable to process prApe1 due to contrast, in typical vps mutants such as vps5, Prc1 remains as precursor, and the majority is secreted into the extracellular its defect in autophagosome formation. Similar to the apg1 strain, the mon1 and ccz1 strains retained the precursor fraction as the p2 (Golgi-modified precursor) form. In the mon1 strain, a small amount of mPrc1 (5%) was found in the form of Ape1 in starvation conditions, suggesting that these two proteins are absolutely required for autophagy. The block intracellular fraction. However, the majority of the protein was found in the p2 form even after 30 min of chase, and approxi- in prApe1 maturation in SD-N was consistent with the starva- tion sensitivity phenotype. Thus, we conclude that Mon1 and mately half was missorted to the extracellular fraction (Fig. 2A). Similar results were seen with Pep4 (data not shown). The Ccz1 are required for both the Cvt and autophagy pathways. We have reported previously that the peroxisome degrada- Prc1-processing defect in the ccz1 strain has already been Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex 47921 FIG.2. Multiple vacuole transport pathways are blocked in the ccz1 and mon1 strains. A, the mon1 strain mis- sorts Prc1 into the extracellular fraction. The wild type (WT, SEY6210), vps5, and mon1 (JSY1) strains were grown to mid- log phase and converted into spheroplasts. The spheroplasts were labeled for 5 min and subjected to a non-radioactive chase for the time indicated at 30 °C. Samples were separated into intracellular (I) and extracellular (E) fractions, immunoprecipi- tated with antiserum to Prc1, and sepa- rated by SDS-PAGE. B, mon1 and ccz1 strains accumulate precursor Pho8. The wild type, pep4 (TVY1), ccz1 (CWY3), and mon1 strains were grown to mid- log phase in SMD medium and analyzed by immunoblot using antiserum against Pho8. C, GFP-Pho8 reaches the vacuoles of the ccz1 and mon1 strains. Wild type, ccz1, mon1, and vam3 (CWY40) strains were transformed with pGFP-Pho8 (426) and grown in SMD medium to mid- log phase followed by fluorescence micros- copy. D, endocytic and MVB vesicles accu- mulated in the ccz1 and mon1 cells outside of their vacuoles. The wild type, ccz1, and mon1 strains expressing the endocytosis pathway marker Ste3-GFP (316), or the MVB pathway marker Sna3 GFP(416) were grown to mid-log phase fol- lowed by fluorescence microscopy. DIC, dif- ferential interference contrast. published (11). Consistent with the published data, the ccz1 and MVB pathways. We monitored endocytosis by looking at strain showed a Prc1 sorting defect by pulse/chase analysis but the localization of Ste3-GFP in the mon1 and ccz1 strains. accumulated a substantial amount of mPrc1 under steady- Ste3 is the a factor receptor and is down-regulated by both state conditions (data not shown). The steady-state accumula- ligand-dependent and ligand-independent modes of endocyto- tion of mPrc1 probably reflects a block in exit from a pre- sis (35). In this study, we examined the ligand-independent vacuolar compartment that has attained protease-processing mode. In the wild type strain, Ste3-GFP was diffusely accumu- capacity (34). lated in the vacuole (Fig. 2D). In contrast, Ste3-GFP was local- Next, we examined the delivery of the vacuole integral mem- ized to multiple punctate structures outside of the vacuole in the mon1 and ccz1 strains. These structures may represent brane protein Pho8 through the ALP pathway. Under steady- state conditions, both the mon1 and ccz1 strains showed an endocytic vesicles. These data indicate an endocytic defect in the mon1 and ccz1 strains. Finally, we examined the local- 50% block of Pho8 processing, while the wild type strain accumulated mature Pho8 (Fig. 2B and Ref. 11). To further ization of Sna3-GFP through the MVB pathway (24). In con- trast to the vacuole lumen staining seen in the wild type cells, examine the delivery of Pho8 in these two mutant strains, we followed the localization of GFP-Pho8. We used a vam3 strain Sna3-GFP in the ccz1 and mon1 cells displayed a large population of small punctate structures outside of vacuoles as a control because the v-SNARE Vam3 is required for the ALP pathway. Wild type, ccz1, mon1, and vam3 strains (Fig. 2D), which may represent the late endosome/MVB com- partments. Similar results were seen using other MVB path- expressing GFP-Pho8 were grown to mid-log phase and exam- ined by fluorescent microscopy. Similar to the severe vacuole way marker proteins including Phm5-GFP and GFP-CPS (data not shown). fragmentation in the vam3 strain, both ccz1 and mon1 also displayed a fragmented vacuole phenotype, although a sub- Ccz1 and Mon1 Are Required for Vesicle Fusion with the Vacuole—The majority of cvt, apg, and aut mutants identified stantial population of cells exhibited some relatively larger vacuoles (Fig. 2, C and D). In wild type cells, GFP-Pho8 was previously were specific to the Cvt and autophagy pathways and did not show defects in other vacuole delivery pathways. detected at the vacuole membrane indicating proper delivery of this hydrolase to the vacuole. In contrast to the wild type cells, These mutants all appear to function at the stage of vesicle induction and/or formation. However, the cvt4 and cvt8 mu- GFP-Pho8 accumulated in multiple punctate structures in the vam3 strain (Fig. 2C). Although vacuoles in the vam3 strain tants were found to be allelic with VPS39/VAM6 and VPS41/ VAM2, respectively (25), indicating a possible overlap with are highly fragmented, we were able to conclude that none of these fluorescent dots were inside of the fragmented vacuoles. genes whose products play a more general role in vacuole protein localization. Because the mon1 and ccz1 mutants are We found that both the ccz1 and mon1 strains accumulated GFP-Pho8 on the vacuole membrane but also displayed some defective in multiple vacuole delivery pathways, we propose that Mon1 and Ccz1 have general roles for protein trafficking punctate GFP-Pho8 dots outside of their fragmented vacuoles, suggesting only a partial block in the delivery of Pho8 (Fig. 2C). pathways presumably through their requirements for the ves- icle fusion step with the vacuole. Similar results were observed by examining cells expressing Nyv1-GFP, which is also delivered to the vacuole by the ALP To carefully examine the proposed role of Ccz1 and Mon1 for the fusion of vesicles with the vacuole, we utilized biochemical pathway (data not shown). These data suggest a partial block in the ALP pathway in the mon1 and ccz1 strains. assays that monitor the block in the transport of prApe1 (17). To determine whether prApe1 was able to bind membrane, we In addition to the Cvt/Apg, CPY, and ALP pathways, pro- teins destined for the vacuole also transit through the endocytic performed a flotation analysis. A total membrane fraction from 47922 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex FIG.3. Mon1 and Ccz1 are required after completion of Cvt vesicles. A, pre- cursor Ape1 is membrane associated in the mon1 strain. The mon1 (JSY1) strain was grown to mid-log phase and converted into spheroplasts. The spheroplasts were lysed osmotically and centrifuged through a Ficoll step gradient with or without Triton X-100 as described under “Experimental Proce- dures.” Membrane-containing float (F), non- float (NF), and pellet (P2) fractions were col- lected and subjected to immunoblot using antisera or antibodies to Ape1, Dpm1, and Pgk1. B, precursor Ape1 is protease-pro- tected in the mon1 and ccz1 strains. The apg7 (VDY101), ypt7 (WSY99), mon1, and ccz1 (CWY3) strains were grown to mid-log phase and converted into sphero- plasts followed by osmotic lysis. The total lysate (T) was resolved into supernatant (S) and pellet (P) fractions by a 13,000 g cen- trifugation, and a portion analyzed by im- munoblot using antiserum to Ape1 and Pgk1. The remaining pellet fractions were subjected to protease treatment in the ab- sence or presence of Triton X-100 and sub- jected to immunoblot using antiserum to Ape1. C, Cvt pathway marker GFP-Aut7 ac- cumulated outside of the vacuole in the mon1 and ccz1 strains. The wild type, ccz1, and mon1 strains were transformed with pCuGFPAut7 (22). The strains were grown to mid-log phase, and images were taken with a fluorescent microscope. lysed spheroplasts was subjected to centrifugation through a contrast, GFP-Aut7 in the mon1 and ccz1 strains displayed Ficoll step gradient. In the mon1 strain, a portion of prApe1 multiple punctate dots similar to that seen in ypt7 cells (Fig. and the integral ER membrane control protein Dpm1 were 3C and Ref. 39). By overlaying the fluorescent and DIC images, pelletable and separated into the float (F) fraction in the ab- we could determine that the multiple punctate structures in sence of detergent (Fig. 3A). In contrast, the cytosolic protein these two strains were located outside of the fragmented vacu- Pgk1 was found exclusively in the supernatant (S) fraction. A oles. Under starvation conditions, we detected a stronger GFP- similar result was seen with the ccz1 strain (data not shown). Aut7 signal in the two mutant strains suggesting that they are This result suggests that prApe1 is able to bind to its target not defective in Aut7 induction. Some larger double membrane membrane. structures that might represent autophagosomes were detected To determine if prApe1 is sequestered within completed Cvt outside of vacuoles in the two mutant strains but none of the GFP-Aut7 appeared to be coincident with the vacuole. Overall, vesicles, we next carried out a protease-sensitivity analysis. Spheroplasts were osmotically lysed as described under “Ex- these data suggest that prApe1 is accumulated within com- pleted cytosolic vesicles in both the mon1 and ccz1 strains. perimental Procedures,” and the low speed pellet fractions were subjected to exogenous proteinase K treatment in the Thus, we conclude that Ccz1 and Mon1 are required for the fusion step of these vesicles with the vacuole. absence or presence of detergent. The apg7 strain is defective in the conjugation of Apg12 to Apg5 and is unable to form Ccz1 and Mon1 Are Membrane-associated Proteins—In order to study the localization of Ccz1 and Mon1, we tagged both completed Cvt vesicles/autophagosomes (36, 37). This strain accumulates prApe1 in a protease-sensitive state in the ab- proteins with the HA epitope. The COOH-terminal HA tagging did not cause dysfunction of Ccz1 or Mon1, because the respec- sence of detergent (Fig. 3B). Ypt7 is a Rab protein that is required for the fusion of Cvt vesicles/autophagosomes with the tive constructs on plasmids complemented the prApe1-sorting defect of null cells and rescued the fragmented vacuole pheno- vacuole (37), and ypt7 cells accumulate protease-protected prApe1. Precursor Ape1 in the mon1 and ccz1 strains was types (data not shown). It has been shown that Ccz1 is enriched in the P13 and P100 fractions (11). We decided to check also protease-protected in the absence of detergent (Fig. 3B), suggesting that it accumulated within completed vesicles. The Mon1-HA localization together with Ccz1-HA. A strain with Mon1-HA tagged at the chromosomal locus was transformed separation of Pgk1 into the supernatant fraction verifies that accumulation of protease-protected prApe1 was not due to in- with pCCZ1-HA(416), grown to mid-log phase, and converted to spheroplasts followed by osmotic lysis. The lysed spheroplasts efficient spheroplast lysis. To determine whether prApe1 was present within cytosolic were subjected to velocity sedimentation as described under “Experimental Procedures.” The cytosolic protein Pgk1 was or subvacuolar vesicles, we extended our analysis of the Cvt pathway by looking at GFP-Aut7 in vivo. Aut7 is required for recovered primarily from the S100 fraction, while the vacuole membrane protein Pho8 was located exclusively in the P13 Cvt vesicle and autophagosome formation and remains associ- ated with these vesicles following completion (22, 38). Thus, it fraction indicating efficient separation (Fig. 4A). We also ex- amined the localization of Ypt7 and found it was mostly in the serves as a vesicle marker. Consistent with previously pub- lished data, GFP-Aut7 was seen as a single punctate structure P13 fraction. Mon1-HA was recovered in the P13 and P100 fractions; however, we found that Ccz1-HA was not only de- accumulating outside the vacuole in the wild type cells grown in rich medium (Fig. 3C, SMD). Under starvation conditions, tected in the P13 and P100 fractions but that a substantial amount also appeared in the S100 fraction indicating a cytoso- Aut7 is induced, and we observed a bright vacuole lumen stain- ing of GFP-Aut7 in the wild type strain (Fig. 3C, SD-N). In lic population of this protein (Fig. 4A). Next we extended our Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex 47923 FIG.4. Ccz1 and Mon1 are peripheral membrane proteins. A, Ccz1-HA and Mon1-HA are pelletable. A strain with an HA tag at the Mon1 locus (PSY35) transformed with pCCZ1-HA(416) was grown to mid-log phase and converted into spheroplasts, followed by osmotic lysis in PS200 buffer containing 5 mM MgCl . The total (T) fraction was separated into low speed supernatant (S13) and pellet (P13) fractions by a 13,000 g centrifugation step. The S13 fraction was further separated into high-speed supernatant (S100) and pellet (P100) frac- tions by centrifugation at 100,000 g. The collected fractions were subjected to immunoblot using antisera to HA, Pgk1, Ypt7, and Pho8. The asterisk marks a cross-reacting band that migrates below Pho8. B, biochemical characterization of pelletable Ccz1-HA and Mon1-HA. Spheroplasts from the Mon1-HA and Ccz1-HA (PSY36) strains were osmotically lysed and spun as described under “Experimental Proce- dures.” The pellet fractions were resuspended in buffer alone or buffer containing 1 M KCl, 0.1 M Na CO , pH 10.5, 3 M urea, or 1% Triton X-100 2 3 and separated into supernatant (S) and pellet (P) fractions. Samples were resolved by immunoblot with anti-HA antiserum. analyses for these two proteins by examining the stability of their membrane binding. We found that both Ccz1-HA and Mon1-HA were largely stripped from the membrane by treat- ment with 0.1 M Na CO (pH 10.5) and 3 M urea, while approx- 2 3 imately half of each protein remained membrane bound in the presence of 1 M KCl and 1% Triton X-100 (Fig. 4B). Taken FIG.5. In vivo localization of Ccz1 and Mon1. Yeast strains with Ccz1-GFP (PSY46) and Mon1-GFP (PSY47) integrated at the chromo- together, these data suggest that Ccz1-HA and Mon1-HA are somal loci were grown to mid-log phase in YPD, then washed and peripherally attached to a membrane compartment(s) that are resuspended in SMD medium (A)orH O (B) before being examined by relatively detergent insoluble. The lack of solubility in the fluorescence microscopy. Ccz1 and Mon1 localize to punctate perivacu- presence of detergent may indicate that both proteins associate olar structures and osmotic shock results in a redistribution to the with a large protein complex. vacuolar rim. DIC, differential interference contrast. C, a yeast strain with chromosomal Ccz1-GFP (PSY46) was grown in YPD to mid-log In Vivo Localization of Ccz1-GFP and GFP-Mon1—To inves- phase, washed, and resuspended in water for 5 min, followed by a shift tigate the site of action of Ccz1 and Mon1 in vivo, we con- to SMD conditions prior to fluorescence microscopy. Images were taken structed strains where GFP was fused to the COOH terminus at minute intervals after the SMD treatment as indicated. Ccz1-GFP of the MON1 and CCZ1 ORFs at the chromosomal loci. These gradually redistributed to the punctate structures within 5 min based on time-lapse microscopy. The vacuolar rim staining is difficult to detect strains displayed a normal vacuolar phenotype indicating that due to photobleaching resulting from the time-lapse exposures. Essen- the expressed fusion proteins are functional (Fig. 5). When cells tially identical results were obtained for Mon1-GFP. grown in YPD to mid-log phase (and washed in minimal me- dium) were examined, Ccz1-GFP was detected in 2–5 perivacu- olar dots per cell and also displayed a faint vacuole membrane staining pattern along with the fainter vacuole ring localization staining (Fig. 5A). These GFP-staining dot structures were of Ccz1-GFP and Mon1-GFP (Fig. 5C). The punctate pattern very mobile and could be seen to move around the vacuoles appeared rapidly and became saturated within 5 min of revers- (data not shown). Mon1-GFP had a similar staining pattern to ing the osmotic conditions. Therefore, we conclude that the Ccz1-GFP, although the fluorescent signal was weaker. majority of Ccz1-Mon1 complexes localize to several membrane Washing yeast cells under low osmotic conditions causes structures right next to the vacuole and could possibly attach to multilobed vacuoles to fuse together and swell. When the Ccz1- the vacuole membrane to achieve their function. GFP and Mon1-GFP yeast were washed with water prior to Because both proteins displayed a similar subcellular distri- microscopy, the vacuoles could be seen to enlarge (Fig. 5B). The bution by fluorescent microscopy, we extended the analysis by punctate staining pattern of Ccz1-GFP and Mon1-GFP was examining the co-localization of YFP- and CFP-tagged pro- largely lost and was replaced by an increased signal on the teins. Because Mon1 tagged chromosomally at the COOH ter- vacuolar rim. Furthermore, by shifting the hypotonic treat- minus with CFP or YFP showed a very weak fluorescent signal, ment back to SMD, we were able to recover the punctate- we replaced chromosomal MON1 with YFP-MON1 under the 47924 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex FIG.6. Ccz1 and Mon1 co-localize to a perivacuolar compart- ment different from the pre-autophagosomal structure. A, strain PSY45 expressing YFP-Mon1 from the CUP1 promoter and Ccz1-CFP was grown to mid-log phase in YPD. YFP-Mon1 expression was induced with 50 M CuSO for 1 h prior to microscopy. B, strain PSY42 express- ing Ccz1-YFP from the chromosomal loci and Cvt19-CFP from a plas- mid, was grown to mid-log phase in SMD and then for1hin YPD. All cells were washed once in SMD before being examined by fluorescence microscopy. control of the CUP1 promoter. The resulting strain showed normal vacuolar morphology (Fig. 6A). Ccz1-CFP and YFP- Mon1 showed multiple punctate dots similar to the pattern seen with the GFP-tagged constructs. Furthermore, the two proteins co-localized (Fig. 6A). The staining pattern seen with GFP-Mon1 and Ccz1-GFP was different from the single punc- tate structure observed with most Apg/Cvt proteins that local- FIG.7. Ccz1 and Mon1 physically interact. A, Ccz1 and Mon1 ize to the pre-autophagosomal structure (20). To determine co-fractionated but were separated from endomembrane marker pro- whether Mon1 and Ccz1 localized to a distinct compartment, teins by OptiPrep density gradients. The Mon1-HA strain (PSY35) we compared the distribution of Ccz1-YFP to Cvt19-CFP. expressing pCCZ1-HA(416) was analyzed by density gradient separa- tion as described under “Experimental Procedures.” Fractions were Cvt19 is a receptor or adaptor for prApe1 (40) and localizes to subjected to immunoblot using antisera or antibodies to Dpm1 (ER), the pre-autophagosomal structure (20). We found that the Anp1 (Golgi), Pep12 (endosome), Pho8 (vacuole), Ypt7, and HA. B, punctate dots corresponding to Ccz1-YFP did not co-localize Ccz1-HA co-precipitates Mon1 by native immunoprecipitation. Wild with the pre-autophagosomal structure represented by Cvt19- type, ccz1 (CWY3), and mon1 (JSY1) strains were transformed with pCCZ1-HA(426), pMON1(426), and/or pYPT7(424), and were grown to CFP (Fig. 6B). mid-log phase followed by glass bead lysis in HEPES native immuno- Ccz1 and Mon1 Form a Stable Protein Complex—We have precipitation buffer. An aliquot (10 l) of lysate was used as the loading shown that the ccz1 and mon1 strains have similar vacuole control. Lysates were incubated with anti-HA antibody and protein protein transport defects, and that Ccz1 and Mon1 are both A-Sepharose as described under “Experimental Procedures” and sub- jected to immunoblot against HA, Mon1, and Ypt7. pelletable and that their association with membranes has sim- ilar biochemical properties. In addition, both proteins co-local- ized by fluorescent microscopy (Fig. 6A). To further investigate significant effect for the wild type or mutant strains with the subcellular localization of Ccz1-HA and Mon1-HA, we re- regard to prApe1 maturation (data not shown). Cells were solved the membrane compartments on an OptiPrep density lysed with glass beads, and the crude cell lysate was subjected gradient as described under “Experimental Procedures.” After to Western blot as the loading control (Fig. 7B, input). We generated polyclonal antiserum against Mon1 as described un- centrifugation, fractions were collected from the top of the gradient and analyzed by immunoblot. Ccz1-HA and Mon1-HA der “Experimental Procedures.” The antiserum detected a very weak band of 70 kDa in the wild type strain and showed a were both detected in fractions 8 through 13 (Fig. 7A). We compared their distribution with endomembrane markers greatly increased level of this band in cells expressing a mul- ticopy MON1 plasmid (Fig. 7B and data not shown). Cells were Dpm1 (ER), Anp1 (Golgi), Pep12 (endosome), and Pho8 (vacu- ole). All these proteins displayed fractionation patterns that subjected to a native immunoprecipitation with antiserum against HA as described under “Experimental Procedures.” were distinct from Ccz1-HA and Mon1-HA. We also checked the localization of Ypt7 that has been suggested to interact The precipitated immune complexes (affinity isolate) were then subjected to SDS-PAGE and Western blots using antibodies or with Ccz1, and found that a population of these proteins over- lapped in fractions 8 and 9, but that the peaks were distinct antiserum against HA, Mon1, and Ypt7. In the wild type strain containing overexpressed Ccz1-HA, the immune complex (Fig. 7A). To extend this analysis we examined whether Ccz1 physi- pulled down a substantial amount of the chromosomal Mon1 (Fig. 7B). The immunoaffinity signal is specific against Mon1 cally interacts with Mon1, using a co-immunoprecipitation as- say. We expressed a combination of pCCZ1-HA(426), pMON1 because no Mon1 signal was detected in the mon1 strain under the same immunoprecipitation conditions. When Mon1 (426), and pYPT7(424) in several strain backgrounds (Fig. 7B). Overexpression of the respective proteins did not cause any was overexpressed in the absence of Ccz1-HA, none of the Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex 47925 FIG.8. Working model for the Cvt and autophagy pathways. The type of vesicles that are produced depends on the nutrient conditions. Autophagosomes form during autophagy under conditions of nutrient deprivation. Cvt vesicles are generated through the Cvt pathway un- der nutrient rich conditions. Four general steps of both pathways are indicated be- low the illustration. Components that are required for the Cvt and Apg pathways are indicated based on their putative roles. Mon1 could be detected in the immune complex indicating that appearance of the chimera on fragmented vacuoles in these two the isolation of Mon1 was dependent on Ccz1 (Fig. 7B). The mutants. In contrast, a vam3 strain accumulated a large reverse interaction was also observed when we carried out the population of small vesicles containing GFP-Pho8 that were not immunoprecipitation using antiserum against Mon1 (data not observed on the fragmented vacuoles. Although we do see some shown). It has been published previously that Ccz1-HA co- GFP-Pho8 on intermediate vesicle structures in the ccz1 and immunoprecipitates with Ypt7 (12). However, we were unable mon1 strains, the majority of GFP-Pho8 targeted to their to detect any Ypt7 signal in this experiment. It is possible that fragmented vacuoles (Fig. 2C). Thus, it appears that the ALP the interaction between Ypt7 and Ccz1 (and maybe also Mon1) pathway that bypasses the endosome is relatively unaffected in is transient, whereas the interaction between Ccz1 and Mon1 is the ccz1 and mon1 strains. The partial Pho8 processing abundant and stable. defect might reflect reduced processing capacity of the vacuole resulting from the missorting of Prc1, Pep4, and other hydro- DISCUSSION lases that utilize the CPY pathway. This possibility is sup- Ccz1 and Mon1 Are Required in Multiple Pathways to the ported by the observation of 50% precursor Pho8 in purified Yeast Vacuole—The biosynthetic Cvt pathway that delivers the vacuoles from the ccz1 and mon1 strains (data not shown). precursor form of the vacuolar hydrolase Ape1 from the cyto- Analysis of Ste3-GFP and Sna3-GFP revealed blocks in the plasm to the yeast vacuole is the subject of our investigation. endocytosis and MVB pathways (Fig. 2D). These data indicate We have identified many cvt mutants and found that most of that mon1 and ccz1 have pleiotropic defects in multiple them exhibited an extensive genetic, biochemical, and morpho- vacuole-sorting pathways. logical overlap with apg and aut mutants that are defective in Ccz1 and Mon1 Function at the Vesicle Fusion Step—The the degradative autophagy pathway (reviewed in Refs. 5 and defect in multiple vacuole delivery pathways and the obser- 28). To gain a comprehensive understanding of the Cvt path- vation of vesicle-like transport intermediates accumulated in way, we further identified mutants defective in prApe1 sorting the two mutants suggested that Mon1 and Ccz1 might act at by screening the yeast deletion library. Using this strategy, we the stage of fusion with the vacuole. Taking advantage of the have identified two new proteins, Ccz1 and Mon1, required for established model for the Cvt/Apg pathway (28), we could the Cvt, autophagy and pexophagy pathways (Fig. 1). To fur- assess the role of Mon1 and Ccz1 through biochemical anal- ther understand the precise role(s) of these two proteins we yses that examined the state of prApe1 (17). In the past few first performed an extensive analysis to determine whether the years, we have dissected the Cvt/Apg pathway into several ccz1 and mon1 strains had pleiotropic effects in other vacu- discrete steps. These include vesicle nucleation and cargo ole transport pathways. The CPY pathway involves transport sequestration, vesicle formation/completion, docking/fusion, through a portion of the secretory pathway, and its itinerary and subvacuolar vesicle lysis followed by maturation of includes the prevacuolar compartment (PVC)/endosome. We prApe1 (Fig. 8). Accordingly, we have developed biochemical found that the CPY pathway cargo protein Prc1 was missorted tools to assess the stage at which the cargo protein prApe1 as the p2 form in the mon1 strain. A similar secretion pheno- accumulates during transport in mutant strains. The mem- type has been shown previously for the ccz1 strain (11). brane flotation analysis indicated that prApe1 in the mon1 The ALP pathway diverges from the CPY pathway in the late strain was membrane-associated (Fig. 3A). Furthermore, we Golgi; cargo proteins such as Pho8 do not pass through the PVC before reaching the vacuole. We showed a partial defect in Pho8 found that prApe1 in the mon1 and ccz1 strains was in a protease-protected form, suggesting that the sequestration processing in these two mutant strains (Fig. 2B). When we further studied the localization of GFP-Pho8, we observed an step for the Cvt complex was completed. Thus, mon1 and 47926 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex ccz1 are the first two mutants that have been isolated in In this study we introduce the novel Ccz1-Mon1 complex as several screens for apg, aut, and cvt mutants that act after acting at the fusion step in the Apg/Cvt pathways, as well as in completion of the sequestering vesicles. most other pathways that involve vesicle fusion with the vac- Protease-protected prApe1 could accumulate in subvacuolar uole. Because of its apparently general role in vacuole biogen- vesicles within the vacuole lumen in strains defective in the esis and function, it is important to examine whether the Ccz1- vesicle lysis step. To verify that the ccz1 and mon1 strains Mon1 complex is part of the basic vacuole fusion mechanism. are defective in delivery to the vacuole, we investigated the For example, what is the specific molecular role of the Ccz1- distribution of GFP-Aut7 in the two mutant strains. Aut7 is a Mon1 complex? Ccz1 has been reported to interact with Ypt7 component that is required for Cvt vesicle formation and is (12). Is the Ccz1-Mon1 complex also part of the Ypt7 effector itself localized to Cvt vesicles (41). Accordingly, Aut7 serves as complex? Although our co-immunoprecipitation data did not a useful vesicle marker. In contrast to the single perivacuolar reproduce the published result of Ccz1-HA and Ypt7 interac- (SMD) or luminal (SD-N) dot observed in the wild type strain, tion, we suggest that this interaction is transient whereas the GFP-Aut7 is localized outside of the vacuole in multiple punc- Ccz1-Mon1 complex is very abundant and stable. We are cur- tate structures in the ccz1 and mon1 strains (Fig. 3C). Fur- rently trying to determine the specific role of the Ccz1-Mon1 thermore, the localization of GFP-Aut7 in the two mutants was complex in the Cvt and Apg pathways. An in vitro analysis will very similar to that seen in the ypt7 strain. Ypt7 is a Rab provide additional insight into their function in the mechanism GTPase that is required for the fusion of multiple vesicle types, of vesicle fusion. including Cvt vesicles and autophagosomes, with the vacuole Acknowledgments—We thank Drs. Scott Emr, Mark Longtine, Sean (37). Taken together, our data indicate that Ccz1 and Mon1 Munro, Jeremy Thorner, and William Wickner and the Yeast Resource function at the stage of fusion of autophagosomes/Cvt vesicles Center for supplying antiserum and plasmids. We thank members of with the vacuole. the Klionsky laboratory, especially Drs. Fulvio Reggiori and John Kim, for helpful discussions and providing plasmids. Molecular Function of Ccz1 and Mon1 in Fusion—Ccz1 and Mon1 are both peripheral membrane proteins (Fig. 4). There is REFERENCES an additional cytosolic pool of Ccz1 (Fig. 4A). The two proteins 1. Klionsky, D. J., Herman, P. K., and Emr, S. D. (1990) Microbiol. Rev. 54, co-localized to a relatively higher density compartment that did 266 –292 not peak at the same position as most endomembrane markers 2. Kim, J., Scott, S. V., and Klionsky, D. J. (2000) Int. Rev. Cytol. 198, 153–201 3. Lemmon, S. K., and Traub, L. M. (2000) Curr. Opin. Cell Biol. 12, 457– 466 (Fig. 7A). Both proteins appeared to function as a stable protein 4. Weisman, L. S., and Wickner, W. (1988) Science 241, 589 –591 complex (Fig. 7B) termed the Ccz1-Mon1 complex in this study. 5. Klionsky, D. J., and Ohsumi, Y. (1999) Annu. Rev. Cell Dev. Biol. 15, 1–32 Similarly, in vivo examination of Ccz1 and Mon1 tagged with 6. Klionsky, D. J. (1998) J. Biol. Chem. 273, 10807–10810 7. Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., and Ohsumi, Y. (1997) J. Cell fluorescent markers suggested that the Ccz1-Mon1 complex Biol. 139, 1687–1695 localizes to perivacuolar dot structures that overlap (Fig. 6). 8. Scott, S. V., Baba, M., Ohsumi, Y., and Klionsky, D. J. (1997) J. Cell Biol. 138, 37– 44 The previously published immunofluorescent data suggest that 9. Klionsky, D. J., and Emr, S. D. (2000) Science 290, 1717–1721 Ccz1 co-localizes with the endosomal marker Nhx1 (11), sug- 10. Harding, T. M., Hefner-Gravink, A., Thumm, M., and Klionsky, D. J. (1996) gesting that these dots might represent some kind of endosome/ J. Biol. Chem. 271, 17621–17624 11. Kucharczyk, R., Dupre, S., Avaro, S., Haguenauer-Tsapis, R., Slonimski, P. P., PVC structure. Neither protein localized to the pre-autophago- and Rytka, J. (2000) J. Cell Sci. 113, 4301– 4311 somal structure that is thought to be the site of Cvt vesicle/ 12. Kucharczyk, R., Kierzek, A. M., Slonimski, P. P., and Rytka, J. (2001) J. Cell Sci. 114, 3137–3145 autophagosome synthesis or the site of the donor membrane 13. Muren, E., Oyen, M., Barmark, G., and Ronne, H. (2001) Yeast 18, 163–172 (Fig. 6). We have also observed faint vacuole membrane stain- 14. Bahler, J., Wu, J. Q., Longtine, M. S., Shah, N. G., McKenzie, A., III, Steever, ing for both Mon1 and Ccz1, indicating these proteins might A. B., Wach, A., Philippsen, P., and Pringle, J. R. (1998) Yeast 14, 943–951 15. Klionsky, D. J., Cueva, R., and Yaver, D. S. (1992) J. Cell Biol. 119, 287–299 target to the vacuole membrane to achieve their function in 16. Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. Biol. 8, fusion. Although we did not observe a vacuole peak of Ccz1 2105–2116 17. Wang, C.-W., Kim, J., Huang, W.-P., Abeliovich, H., Stromhaug, P. E., Dunn, and/or Mon1 in our OptiPrep density gradient, we could not W. A., Jr., and Klionsky, D. J. (2001) J. Biol. Chem. 276, 30442–30451 rule out the possibility that their association with the vacuole 18. Noda, T., Kim, J., Huang, W.-P., Baba, M., Tokunaga, C., Ohsumi, Y., and is relatively weak and is lost during the biochemical proce- Klionsky, D. J. (2000) J. Cell Biol. 148, 465– 480 19. Rehling, P., Darsow, T., Katzmann, D. J., and Emr, S. D. (1999) Nat. Cell Biol. dures. A similar phenotype is seen with Cvt18, which is lost 1, 346 –353 from the vacuole following spheroplast lysis (42). 20. Kim, J., Huang, W.-P., Stromhaug, P. E., and Klionsky, D. J. (2002) J. Biol. Chem. 277, 763–773 A summary of the published data of the components involved 21. Urbanowski, J. L., and Piper, R. C. (1999) J. Biol. Chem. 274, 38061–38070 in the Cvt/Apg pathways is shown in Fig. 8. At the fusion step, 22. Kim, J., Huang, W.-P., and Klionsky, D. J. (2001) J. Cell Biol. 152, 51– 64 SNARE proteins including Vam3 (43), Vti1 (44), and Vam7 (45) 23. Cowles, C. R., Snyder, W. B., Burd, C. G., and Emr, S. D. (1997) EMBO J. 16, 2769 –2782 are required for both Cvt vesicle and autophagosome fusion 24. Reggiori, F., and Pelham, H. R. B. (2001) EMBO J. 20, 5176 –5186 with the vacuole. Ypt7, the Rab GTPase (37), and its proposed 25. Harding, T. M., Morano, K. A., Scott, S. V., and Klionsky, D. J. (1995) J. Cell Biol. 131, 591– 602 effector complex, termed the class C Vps/HOPS (homotypic 26. Kim, J., Kamada, Y., Stromhaug, P. E., Guan, J., Hefner-Gravink, A., Baba, fusion and vacuole protein sorting) complex that comprises M., Scott, S. V., Ohsumi, Y., Dunn, W. A., Jr., and Klionsky, D. J. (2001) Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41 (25, 46), is also J. Cell Biol. 153, 381–396 27. Vida, T. A., and Emr, S. D. (1995) J. Cell Biol. 128, 779 –792 essential machinery at this step. Among these identified com- 28. Kim, J., and Klionsky, D. J. (2000) Annu. Rev. Biochem. 69, 303–342 ponents required for the Cvt vesicle/autophagosome fusion 29. Kucharczyk, R., Gromadka, R., Migdalski, A., Slonimski, P. P., and Rytka, J. (1999) Yeast 15, 987–1000 step, none retain a normal vacuole phenotype when they are 30. Abeliovich, H., Dunn, W. A., Jr., Kim, J., and Klionsky, D. J. (2000) J. Cell Biol. deleted from the genome. On the other hand, most but not all 151, 1025–1034 mutants that exhibit a vacuole fragmentation phenotype are 31. 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. defective for the Cvt/autophagy pathway. For example, al- (2000) J. Biol. Chem. 275, 25840 –25849 though the kcs1 strain showed a fragmented vacuole pheno- 32. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., and type, it accumulated the mature form of Ape1 (Ref. 47). Ohsumi, Y. (2000) J. Cell Biol. 150, 1507–1513 Sim- 33. Hutchins, M. U., Veenhuis, M., and Klionsky, D. J. (1999) J. Cell Sci. 112, ilarly, some vps mutants such as vps5 have fragmented 4079 – 4087 vacuoles but are essentially normal for import of prApe1 (15). 34. Piper, R. C., Cooper, A. A., Yang, H., and Stevens, T. H. (1995) J. Cell Biol. 131, 603– 617 35. Chen, L., and Davis, N. G. (2000) J. Cell Biol. 151, 731–738 36. Tanida, I., Mizushima, N., Kiyooka, M., Ohsumi, M., Ueno, T., Ohsumi, Y., and P. E. Stromhaug and D. J. Klionsky, unpublished data. Kominami, E. (1999) Mol. Biol. Cell 10, 1367–1379 Vesicle Fusion with the Vacuole Requires the Ccz1-Mon1 Complex 47927 37. Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., and Klionsky, D. J. (1999) 43. Darsow, T., Rieder, S. E., and Emr, S. D. (1997) J. Cell Biol. 138, 517–529 Mol. Biol. Cell 10, 1337–1351 44. Fischer von Mollard, G., and Stevens, T. H. (1999) Mol. Biol. Cell 10, 38. Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, 1719 –1732 T., Ohsumi, M., Takao, T., Noda, T., and Ohsumi, Y. (2000) J. Cell Biol. 151, 45. Sato, T. K., Darsow, T., and Emr, S. D. (1998) Mol. Cell. Biol. 18, 5308 –5319 263–276 46. Sato, T. K., Rehling, P., Peterson, M. R., and Emr, S. D. (2000) Mol. Cell 6, 39. Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T., and Ohsumi, Y. 661– 671 (2001) EMBO J. 20, 5971–5981 47. Seeley, E. S., Kato, M., Margolis, N., Wickner, W., and Eitzen, G. (2002) Mol. 40. Scott, S. V., Guan, J., Hutchins, M. U., Kim, J., and Klionsky, D. J. (2001) Mol. Biol. Cell 13, 782–794 Cell 7, 1131–1141 48. Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. 41. Huang, W.-P., Scott, S. V., Kim, J., and Klionsky, D. J. (2000) J. Biol. Chem. Biol. 8, 4936 – 4948 275, 5845–5851 49. Wurmser, A. E., and Emr, S. D. (1998) EMBO J. 17, 4930 – 4942 42. Guan, J., Stromhaug, P. E., George, M. D., Habibzadegah-Tari, P., Bevan, A., 50. Gerhardt, B., Kordas, T. J., Thompson, C. M., Patel, P., and Vida, T. (1998) Dunn, W. A., Jr., and Klionsky, D. J. (2001) Mol. Biol. Cell 12, 3821–3838 J. Biol. Chem. 273, 15818 –15829
Journal
Journal of Biological Chemistry – Unpaywall
Published: Dec 1, 2002