The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m208191200
ISSN1083-351X
AutoresChao-Wen Wang, Per E. Strømhaug, Jun Shima, Daniel J. Klionsky,
Tópico(s)Peptidase Inhibition and Analysis
ResumoMon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ andmon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion. Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ andmon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion. Compartmentalization allows eukaryotic cells to regulate intracellular functions by separating competing reactions and localizing enzymes and substrates at specific locations within the cell. Efficient compartmentalization necessitates dynamic protein trafficking processes by which cells are able to establish and maintain the identity and function of each organelle. The vacuole (lysosome) of the yeast Saccharomyces cerevisiae plays a central role in the turnover of cytoplasmic organelles, degradation of intracellular/extracellular components, and maintenance of cellular physiology (1Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Google Scholar). To carry out these functions, the vacuole maintains a variety of degradative enzymes. Both resident hydrolases and their substrates arrive at this destination through a variety of sorting pathways. The main routes by which vacuolar hydrolases are delivered to this organelle are the carboxypeptidase Y (CPY), 1The abbreviations used are: CPY, carboxypeptidase Y; ALP, alkaline phosphatase; Ape1, aminopeptidase I; CFP, cyan fluorescent protein; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent protein; prApe1, precursor aminopeptidase I; PVC, pre-vacuolar compartment; SMD, synthetic minimal medium with dextrose; SD/-N, synthetic minimal medium with dextrose but lacking nitrogen; YFP, yellow fluorescent protein; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid alkaline phosphatase (ALP), and multivesicular body (MVB) pathways, which involve transit through a portion of the secretory pathway, and the cytoplasm to vacuole targeting (Cvt) pathway by which the cargo molecules are packaged as cytosolic membrane-bound intermediates (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 3Lemmon S.K. Traub L.M. Curr. Opin. Cell Biol. 2000; 12: 457-466Google Scholar). Resident proteins are also transmitted by inheritance from mother cell vacuoles to daughter cells during cell division (4Weisman L.S. Wickner W. Science. 1988; 241: 589-591Google Scholar). Substrates enter the vacuole through endocytosis, autophagy and the vacuole import and degradation pathway (reviewed in Ref. 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar). One common feature in all of these processes is membrane fusion. The membrane fusion mechanism acts to ensure specificity for the directed movement of proteins while also maintaining the distinct composition of each organelle within the highly compartmentalized eukaryotic cell. The cytoplasm to vacuole targeting pathway that is used to deliver the soluble hydrolase aminopeptidase I (Ape1) to the vacuole has been under investigation (for reviews see Refs. 2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar, and 6Klionsky D.J. J. Biol. Chem. 1998; 273: 10807-10810Google Scholar). Under vegetative conditions, precursor Ape1 (prApe1) is assembled into a large Cvt complex composed in part of multiple prApe1 dodecamers in the cytosol that becomes enwrapped within a double-membrane Cvt vesicle (7Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Google Scholar). Upon completion, the cytosolic Cvt vesicle targets to the vacuole. The outer membrane of the Cvt vesicle fuses with the vacuole membrane and the intact inner vesicle (Cvt body) passes into the vacuole lumen (8Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Google Scholar). The Cvt body is ultimately broken down by resident vacuolar hydrolases, resulting in the release and maturation of prApe1. Precursor Ape1 is transported to the vacuole by another pathway, termed autophagy (Apg), under starvation conditions (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 9Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Google Scholar). In the Apg pathway, portions of cytoplasm are sequestered within relatively larger double membrane vesicles (autophagosomes) that are also targeted to the vacuole (7Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Google Scholar). Although Apg is a degradative process, mutants defective in autophagy,apg/aut, overlap with cvt mutants (10Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Google Scholar). Morphological and biochemical analyses further indicate that the Cvt and Apg pathways use analogous mechanisms (2Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Google Scholar, 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar, 9Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Google Scholar). 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 for Cvt/Apg import that had not been previously implicated in these pathways. The product of one of these genes, Ccz1, has been suggested to be involved in multiple trafficking pathways to the vacuole (11Kucharczyk R. Dupre S. Avaro S. Haguenauer-Tsapis R. Slonimski P.P. Rytka J. J. Cell Sci. 2000; 113: 4301-4311Google Scholar). Overexpression of the Rab protein Ypt7 rescues the sensitivity to calcium, caffeine, and zinc observed with the ccz1Δ strain. The Ypt7K127E mutant has been identified as a specific mutation that suppresses the ccz1Δ phenotype (12Kucharczyk R. Kierzek A.M. Slonimski P.P. Rytka J. J. Cell Sci. 2001; 114: 3137-3145Google Scholar). Co-immunoprecipitation data further support the physical interaction between Ccz1 and Ypt7 (12Kucharczyk R. Kierzek A.M. Slonimski P.P. Rytka J. J. Cell Sci. 2001; 114: 3137-3145Google Scholar). The mon1Δ strain is sensitive to monensin and brefeldin A (13Muren E. Oyen M. Barmark G. Ronne H. Yeast. 2001; 18: 163-172Google Scholar), but is otherwise uncharacterized. In this study, we show that strains lacking either of these two proteins have similar phenotypes. Both Mon1 and Ccz1 are required not only for the Cvt/Apg pathways but also other vacuole biogenesis processes including the sorting of newly synthesized vacuolar proteins through the CPY, ALP, and MVB pathways and endocytosis. Biochemical and morphological evidence further indicate that the Cvt/Apg pathways are blocked at a stage after the formation of the sequestering vesicles but prior to their fusion with the vacuole. These studies also suggest that Ccz1 and Mon1 co-localize to a unique membrane and that they physically interact. Finally, we demonstrate the in vivo localization of these two proteins to a perivacuolar compartment and the vacuole membrane, a site consistent with their proposed role in fusion. The yeast strains used in this study are listed in Table I. Synthetic minimal medium (SMD) contained 0.67% yeast nitrogen base without amino acids, 2% glucose, and auxotrophic amino acids and vitamins as needed. Nitrogen starvation medium (SD-N) contained 0.17% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose. YPD medium contained 1% yeast extract, 2% peptone, and 2% glucose. S. cerevisiae strains were grown at 30 °C. Yeast cells used for this study were grown in the appropriate SMD medium to mid-log (OD600 of 0.6).Table IYeast strains used in this studyStrainGenotypeReferenceSEY6210MATα leu2–3,112 ura3–52 his3–Δ200 trp1–Δ901 lys2–801 suc2–Δ9 GAL(48Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Google Scholar)CWY3SEY6210ccz1Δ::HIS5This studyJSY1SEY6210mon1Δ::HIS5This studyBY4742MATα his3Δ leu2Δ lys2Δ ura3ΔResGenccz1ΔBY4742ccz1Δ::KanMXResGenmon1ΔBY4742mon1Δ::KanMXResGenD3Y102SEY6210 vac8Δ(31Scott S.V. Nice III, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Google Scholar)NNY20MATaura3 trp1 leu2 apg1Δ::LEU2(31Scott S.V. Nice III, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Google Scholar)vps5SEY6210 vps5(48Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Google Scholar)CWY40SEY6210vam3Δ::TRP1This studyWSY99SEY6210ypt7Δ::HIS3(49Wurmser A.E. Emr S.D. EMBO J. 1998; 17: 4930-4942Google Scholar)VDY101SEY6210apg7Δ::LEU2(37Kim J. Dalton V.M. Eggerton K.P. Scott S.V. Klionsky D.J. Mol. Biol. Cell. 1999; 10: 1337-1351Google Scholar)PSY35SEY6210MON1-HA::TRP1This studyPSY36SEY6210CCZ1-HA::TRP1This studyPSY42SEY6210CCZ1-YFP::HIS3This studyPSY44SEY6210CCZ1-CFP::KanMXThis studyPSY45SEY6210 CCZ1-CFP::KanMX pCUP1-YFP-MON1::URA3This studyPSY46SEY6210CCZ1-GFP::HIS3This studyPSY47SEY6210MON1-GFP::HIS3This studyTVY1SEY6210pep4Δ::LEU2(50Gerhardt B. Kordas T.J. Thompson C.M. Patel P. Vida T. J. Biol. Chem. 1998; 273: 15818-15829Google Scholar) Open table in a new tab Reagents for growth medium were from Difco Laboratories (Detroit, MI). DNA restriction enzymes, T4 DNA ligase and calf intestinal alkaline phosphatase were obtained from New England Biolabs, Inc. (Beverly, MA). Tran[35S] label was obtained from ICN (Costa Mesa, CA). Oxalyticase was from Enzogenetics (Corvallis, OR). OptiPrepTM was from Accurate Chemical and Scientific Corp. (Westbury, NY). CompleteTMEDTA-free protease inhibitor was obtained from Roche Molecular Biochemicals. The pME3 vector containing the Schizosaccharomyces pombe HIS5 auxotrophic marker was a gift from Dr. Neta Dean (State University of New York, Stony Brook, NY). The pFA6a knockout and tagging vectors containing TRP1, HIS3, orKanMX markers were generous gifts from Dr. Mark Longtine (Oklahoma State University) (14Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie III, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 943-951Google Scholar). The CFP (pDH3) and YFP (pDH5) plasmids were from the Yeast Resource Center (University of Washington). FM 4-64 dye was obtained from Molecular Probes (Eugene, OR). All other reagents were from Sigma-Aldrich. Antisera against Ape1 (15Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Google Scholar), Prc1 (16Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 2105-2116Google Scholar), and Pep4 (16Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 2105-2116Google Scholar) have been described. Antisera against Pgk1, Ypt7, and Anp1 were provided by Dr. Jeremy Thorner (University of California, Berkeley, CA), Dr. William Wickner (Dartmouth Medical School, Hanover, NH), and Dr. Sean Munro (MRC Laboratory of Molecular Biology, Cambridge, UK), respectively. Antibodies against Pho8, Dpm1, and Pep12 were obtained from Molecular Probes, and the anti-HA antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). To prepare antiserum against Mon1, the NH2 terminus of the Mon1 ORF (1–585 bp) was PCR-amplified and fused to the COOH terminus of the maltose-binding protein. The resulting plasmid was transformed into E. colistrain BL21. Fusion protein purification and antiserum generation were as described (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). AMATα haploid gene deletion library was obtained from ResGen/Invitrogen Corporation (Huntsville, AL). The mutants provided from the company were inoculated on YPD plates and incubated at 30 °C for 12–24 h. The cells on YPD plates were collect and resuspended in 50 μl of MURB (50 mm NaPO4, 25 mm MES, pH 7.0, 1% SDS, 3 m urea, 0.5% β-mercaptoethanol, 1 mm NaN3, and 0.05% bromphenol blue) and converted into crude cell extracts by glass bead lysis and boiling. The extracts were subjected to immunoblot analysis using anti-Ape1 antisera. The chromosomal MON1 and CCZ1 loci were deleted by a PCR-based, one-step procedure (18Noda T. Kim J. Huang W.-P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Google Scholar). In brief, the corresponding auxotrophic marker was amplified from the pME3 or pFA6a knockout plasmids by PCR using oligonucleotides that contained sequences outside of the marker, flanked by sequences that encode regions at the beginning and end of the corresponding ORFs. PCR products were used to transform yeast strain SEY6210. Putative knockout strains were checked by Western blot for the Ape1 phenotype. Similar strategies were applied for the chromosomal HA and fluorescent protein tagging. To clone theMON1 and CCZ1 genes, both ORFs and their upstream/downstream sequences were PCR-amplified using genomic DNA as template. The resulting PCR products for MON1 include 360 bp before the sequence encoding the start codon and 405 bp after the stop codon. The fragments were digested with SacI andSmaI and inserted into the SacI andSmaI site of the pRS416/426 vector to generate plasmids pMON1(416/426). The PCR products for the cloning of CCZ1contain ∼300-bp upstream and 700-bp downstream of the CCZ1ORF. The PCR products were digested with KpnI to generate pCCZ1(416/426). To construct COOH-terminal HA epitope-tagged Ccz1, theCCZ1 ORF was PCR-amplified using pCCZ1(416) as a template. The resulting PCR product was digested and inserted into pRS416HA and pRS426HA that contains a 3×HA epitope (19Rehling P. Darsow T. Katzmann D.J. Emr S.D. Nat. Cell Biol. 1999; 1: 346-353Google Scholar). To construct an NH2-terminal YFP fusion to Mon1, the MON1 ORF was PCR-amplified using pMON1 (416) as a template. The resulting PCR products were inserted into pCuYFP (306) to generate pCuYFP-MON1 (306). The construct was linearized with KpnI and transformed into strain PSY44 to replace endogenous MON1 with pCuYFP-MON1 (strain PSY45). The plasmids pCvt19-CFP(414) (20Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Google Scholar), pSte3-GFP(316) (21Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Google Scholar), pCuGFP-Aut7 (416) (22Kim J. Huang W.-P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Google Scholar), pGFP-Pho8(426) (23Cowles C.R. Snyder W.B. Burd C.G. Emr S.D. EMBO J. 1997; 16: 2769-2782Google Scholar), and pSna3-GFP(416) (24Reggiori F. Pelham H.R.B. EMBO J. 2001; 20: 5176-5186Google Scholar) were described previously. All oligonucleotide sequences and additional details of the plasmid constructions will be provided upon request. Immunoblot analysis was carried out essentially as described previously (25Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Google Scholar). For kinetic analysis of Prc1, yeast cells were grown to an OD600 of 1.0 and converted into spheroplasts. The spheroplasts from 20 OD600 units of cells were resuspended in 300 μl of SMD medium containing 1.3m sorbitol, and labeled with 20 μCi of Tran[35S] label for 5 min, followed by a chase reaction in SMD containing 1.3 m sorbitol, 0.2% yeast extract, 4 mm methionine, and 2 mm cysteine at a final density of 2.0 OD600/ml. Samples were removed at the indicated time points and 1 mm NaN3 was added to stop the reaction. The samples were subjected to a 5,000 ×g centrifugation for 3 min. The resulting supernatant and pellet fractions were separately precipitated with 10% trichloroacetic acid. Trichloroacetic acid precipitates were resuspended in MURB buffer and subjected to immunoprecipitation as described previously (25Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Google Scholar). For kinetic analyses of Ape1, Pep4, and Ste3, yeast cells were grown to an OD600 of 1.0 in SMD medium. Cells (20 OD600units) were resuspended in 300 μl of SMD medium and labeled with 20 μCi of Tran[35S] label for 5–10 min, followed by a chase reaction as above at a final density of 20 OD600/ml. Samples were removed at the indicated time points and precipitated with 10% trichloroacetic acid. Crude extracts were prepared by glass bead lysis and subjected to immunoprecipitation as described previously (25Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Google Scholar). Cell viability and starvation curves and peroxisome degradation rates were determined as described previously (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). The membrane flotation assay was performed essentially by the method described previously (26Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Google Scholar) with minor modifications. Spheroplasts derived from the mon1Δstrain were resuspended in PS200 lysis buffer (20 mm PIPES, pH 6.8, 200 mm sorbitol) containing 5 mmMgCl2 at a spheroplast density of 20 OD600/ml. The lysate was centrifuged at 13,000 × g for 5 min at 4 °C. The pellet fractions from 10 OD600 units of cells were resuspended in 100 μl of 15% Ficoll-400 (w/v) in lysis buffer with or without the addition of 0.2% Triton X-100. The resuspended pellet fractions were overlaid with 1 ml of 13% Ficoll-400 in lysis buffer and then overlaid with 200 μl of 2% Ficoll-400 in lysis buffer. The resulting step gradient was subjected to centrifugation at 13,000 × g for 10 min at 4 °C. The top 500 μl was designated as the float fraction (F), the remaining solution was considered as the nonfloat fraction (NF), and the gradient pellet was designated as the pellet fraction (P). The three fractions were trichloroacetic acid-precipitated, washed twice with acetone, and analyzed by immunoblot. The protease protection assay was performed as described previously (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). In brief, log-phase cultures were subjected to osmotic lysis in PS200 containing 5 mmMgCl2. The lysates were centrifuged at 13,000 × g for 10 min, and the pellet fractions (P13) were resuspended in lysis buffer in the presence or absence of 50 μg/ml proteinase K and/or 0.2% Triton X-100. Reactions were carried out on ice for 30 min followed by trichloroacetic acid precipitation and immunoblot analysis. Mon1-HA cells expressing pCcz1-HA(416) were grown to mid-log phase (OD600 = 0.6) in SMD medium. The cells were converted into spheroplasts and resuspended in PS200 lysis buffer containing 5 mm MgCl2 and the CompleteTM EDTA-free protease inhibitor mixture at a density of 20 OD600/ml. After a preclearing spin (500 × g, for 5 min, at 4 °C), the total lysate was subjected to low-speed centrifugation (13,000 × g for 10 min), resulting in the supernatant (S13) and pellet (P13) fractions. The S13 fraction was subjected to high speed centrifugation (100,000 ×g for 30 min at 4 °C) to generate the supernatant (S100) and pellet (P100) fractions. The resulting fractions were subjected to immunoblot analysis. To examine membrane binding of Ccz1-HA and Mon1-HA the membrane fractions from lysed spheroplasts were treated with 1m KCl, 0.1 m Na2CO3 (pH 10.5), 3 m urea, or 1% Triton X-100 as described previously (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). OptiPrep™ density gradient analysis was performed using a modification of a previously described procedure (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). In brief, a Mon1-HA strain expressing Ccz1-HA was grown to mid-log phase (OD600 = 0.6) and converted into spheroplasts. The spheroplasts were subjected to osmotic lysis in PS200 containing 1 mm EDTA, 1 mm MgCl2, and a protease inhibitor mixture (CompleteTM EDTA-free protease inhibitor tablets, 1 μg/ml leupeptin and 1 μg/ml pepstatin A). The lysate was subjected to very low speed centrifugation (800 × gfor 5 min) to remove the remaining intact spheroplasts. After this preclearing step, the crude lysate from 35 OD600 units of cells was centrifuged at 100,000 × g for 20 min at 4 °C. The resulting total membrane fraction was resuspended in 200 μl of lysis buffer and then applied on top of a density gradient (12 ml, linear) consisting of 10–55% OptiPrep™ in PS200 lysis buffer containing 1 mm EDTA, 1 mm MgCl2, 1 mm dithiothreitol, and a protease inhibitor mixture. The gradients were subjected to centrifugation at 100,000 ×g for 12 h at 4 °C in a Sorvall Th-641 rotor. Samples were collected from the top of the gradients into 14 fractions. The fractions were trichloroacetic acid-precipitated and washed twice with acetone followed by immunoblot analyses. The protocol for co-immunoprecipitation with Ccz1-HA was modified from a previously described procedure (12Kucharczyk R. Kierzek A.M. Slonimski P.P. Rytka J. J. Cell Sci. 2001; 114: 3137-3145Google Scholar). In brief, 10 OD600 units of log-phase cells were lysed with glass beads in lysis buffer (50 mm HEPES, pH 7.4, 150 mm KCl, 1 mmEDTA, 0.5% Triton X-100) with the addition of protease inhibitor mixture and 1 mm phenylmethylsulfonyl fluoride. After a 10-min solubilization on ice, total cell lysates were centrifuged at 13,000 × g for 15 min at 4 °C. To the resulting supernatant, 10 μl of anti-HA antiserum was added followed by incubation with protein A-Sepharose at 4 °C overnight. Sepharose beads were washed with lysis buffer a total of eight times. Bound proteins were eluted in MURB followed by SDS-PAGE and Western blot analysis. All strains used for microscopy were grown in SMD medium to mid-log phase. In vivo FM 4–64 staining was performed as described previously (27Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Google Scholar). Microscopy analysis was performed using a Nikon E-800 fluorescent microscope (Mager Scientific Inc., Dexter, MI). Images were captured by an ORCA II CCD camera (Hamamatsu Corp., Bridgewater, NJ) using Openlab 3 software (Improvision, Inc., Lexington, MA). Although various cvt, apg, andaut mutants defective in the Cvt and Apg pathways have been isolated and analyzed (reviewed in Refs. 5Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar and 28Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Google Scholar), many questions concerning these pathways remain to be answered. We are interested in the molecular mechanism governing the dynamic aspects of the Cvt and Apg pathways. We reasoned that the identification of additional mutants would provide further insight into the protein machinery of these processes. Accordingly, we screened a haploid gene deletion library based on the accumulation of prApe1, a cargo protein that is delivered to the vacuole through the Cvt/Apg pathways. Among the new mutants identified, mon1Δ and ccz1Δ showed a complete block in prApe1 maturation. Although mon1Δ has not been previously reported as having a role in the Cvt pathway, complementation analyses indicate that CCZ1 is allelic withCVT16, a previously uncharacterized CVT gene (10Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Google Scholar). The ccz1Δ mutant was originally identified due to its sensitivity to caffeine, calcium, andzinc (29Kucharczyk R. Gromadka R. Migdalski A. Slonimski P.P. Rytka J. Yeast. 1999; 15: 987-1000Google Scholar). It has also been shown that the strain displays a severe vacuole protein-sorting defect. Immunofluorescent data suggest that Ccz1 localizes to the endosomal compartment, and it has been suggested to act in concert with the Rab protein Ypt7 (11Kucharczyk R. Dupre S. Avaro S. Haguenauer-Tsapis R. Slonimski P.P. Rytka J. J. Cell Sci. 2000; 113: 4301-4311Google Scholar, 12Kucharczyk R. Kierzek A.M. Slonimski P.P. Rytka J. J. Cell Sci. 2001; 114: 3137-3145Google Scholar). There has not been a published report describing Mon1 function. TheMON1 gene, YGL124c, encodes a 644-amino acid protein with a predicted molecular mass of 73.5 kDa. A data base search indicates that Mon1 does not have homology with other proteins ofS. cerevisiae. However, possible homologues having 24–37% identity with Mon1 exist in S. pombe,Caenorhabditis elegans, and Drosophila melanogaster. Ccz1 has no significant homologues. When wild type cells are grown under nutrient-rich conditions, the majority of Ape1 is present as the 50-kDa mature form (Fig.1 A), although a small fraction is present as the 61-kDa precursor. In contrast, both themon1Δ and ccz1Δ strains accumulated only the precursor form of Ape1. The defect in prApe1 processing in these mutants was rescued by expressing either single or multicopy versions of the corresponding genes on plasmids, confirming the essential roles of Mon1 and Ccz1 for the Cvt pathway (Fig. 1 A). Precursor Ape1 is delivered to the vacuole through autophagy under starvation conditions. We utilized a starvation-sensitivity analysis to determine whether the mon1Δ and ccz1Δ strains were able to carry out autophagy. Wild type cells, or mutants specific to the Cvt pathway, are starvation-resistant while mutants defective for autophagy lose viability in the absence of nitrogen (17Wang C.-W. Kim J. Huang W.-P. Abeliovich H. Stromhaug P.E. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2001; 276: 30442-30451Google Scholar). As shown in Fig.1 B, the wild type strain was resistant to starvation over the time course examined. In contrast, mon1Δ andccz1Δ strains, similar to the apg1Δ mutant, displayed a rapid loss of viability in SD-N medium. Viability in themon1Δ strain was restored when these cells expressed Mon1 from a CEN-based plasmid. Starvation-sensitivity indicates that autophagy is not fully functional in the mon1Δ and ccz1Δ strains. Recently, however, we have demonstrated that some mutants that are autophagy defective by this criterion are still able to induce the formation of autophagosomes under starvation conditions. For example, theaut7Δ strain is starvation-sensitive but is able to induce the formation of small, abnormal autophagosomes in SD-N (30Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Google Scholar). In addition, some components of the Cvt and Apg pathways are only essential for one of these two pathways. For example, Vac8 and Cvt9 are only required for the Cvt pathway whereas Apg17 appears to function only in autophagy (26Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Google Scholar, 31Scott S.V. Nice III, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Google Scholar, 32Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Google Scholar). Accordingly, these types of mutants are able
Referência(s)