Correspondence to: Daniel J. Klionsky, Department of Biology, University of Michigan, Ann Arbor, MI 48109. Tel:(734) 615-6556 Fax:(734) 647-0884 E-mail:klionsky{at}umich.edu.
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Abstract |
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Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.
We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
Key Words: autophagy, degradation, lysosome, peroxisome, vacuole
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Introduction |
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General cell function and homeostasis require the regulated counterbalancing of protein synthesis and organelle biogenesis with protein breakdown and the selective degradation of organelles. The macroautophagy pathway directs the lysosome-mediated breakdown of bulk cytoplasm, including proteins, nucleic acids, lipids, and organelles and serves as the principal cellular degradative process (for review see
Biochemical and microscopy studies have outlined the basic steps of macroautophagy in Saccharomyces cerevisiae (for review see
Insights into the molecular details of macroautophagy have been greatly facilitated through molecular genetic studies in S. cerevisiae (
The formation and completion of autophagosomes requires the function of a novel Apg protein conjugation system which results in the covalent linkage of Apg12 to Apg5 (
The majority of the components required for starvation-induced macroautophagy are also employed to transport selective cargo. Under nutrient-rich conditions, the cytoplasm to vacuole targeting (Cvt)1 pathway transports the resident hydrolase aminopeptidase I (API) to the vacuole (
In addition to the Cvt pathway, macroautophagy machinery is essential for the selective delivery of excess peroxisomes to the vacuole by the related pexophagy pathway (for review see
A key question that remains to be answered is what differentiates nonselelective bulk cargo from the specifically targeted cargo, such as prAPI and peroxisomes? Indiscriminate packaging of physiologically critical organelles would result in severe cellular dysfunction. On the other hand, maintaining excess organelles would be energetically wasteful, whereas the failure to sequester and degrade nonfunctional or malfunctioning organelles could result in cell death. To provide such specificity, additional factors are likely required to mediate the sequestration of particular cargo.
In this study, we have identified Cvt9 in S. cerevisiae and Gsa9 in P. pastoris, which are structural and functional homologues. Cvt9 was identified by its requirement for vacuole uptake of prAPI, and Gsa9 by its role in the turnover of peroxisomal enzymes. Although both Cvt9 and Gsa9 are required for the selective uptake of peroxisomes into the yeast vacuole for degradation, neither protein is essential for nonselective macroautophagy induced by nitrogen starvation. These proteins are associated with a unique membrane compartment located in a perivacuolar region of the cell. Our biochemical and morphological results suggest that Cvt9 and Gsa9 function to selectively sequester cytoplasmic proteins and organelles for degradation within the vacuole.
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Materials and Methods |
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Strains
The yeast strains used in this study are listed in Table 1.
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Growth Media
S. cerevisiae strains were grown in synthetic minimal medium (SMD: 0.67% yeast nitrogen base [YNB], 2% glucose, and auxotrophic amino acids and vitamins as needed). Starvation experiments were carried out in SD-N (0.17% YNB without ammonium sulfate or amino acids, and 2% glucose). Peroxisomes were induced by growth in oleic acid medium (YTO: 0.67% YNB, 0.1% Tween 40, and 0.1% oleic acid). P. pastoris strains were grown in YPD (1% Bacto yeast extract, 2% Bacto peptone, and 2% glucose). Peroxisomes were induced by growth in methanol (YNM: 0.67% YNB, 0.4 mg/liter biotin, and 0.5% vol/vol methanol). Degradation of peroxisomes was carried out in YND (0.67% YNB, 0.4 mg/liter biotin, and 2% glucose).
Antibodies/Antiserum
Rabbit antisera against Cvt9 peptides corresponding to amino acids 356382 and 534547 (Multiple Peptide Systems) were generated as described previously (
Materials
Reagents are identical to those described previously (
Cloning CVT9
The cvt9 strain AHY96 (
Isolation of gsa9 Mutants and Cloning GSA9
P. pastoris gsa mutants were isolated after the restriction enzymemediated integration of a 2.0-kb pREMI plasmid which contained the Col E1 origin of replication and the Zeocin resistance gene under the control of the TEF1 promoter from S. cerevisiae and the EM7 promoter of Escherichia coli. Details of the mutagenesis procedure will be described elsewhere. Three different gsa mutants had a disruption of the GSA9 gene. gsa9-1 cells had the pREMI vector inserted between Q1068 and Y1069, gsa9-2 between M83 and S84, and gsa9-3 between R1291 and A1292. Using the different genomic DNA fragments from the three different allelic mutants, we were able to completely assemble the GSA9 gene as well as 700 bp of the 5' and 600 bp of the 3' noncoding regions. The genomic sequence of GSA9 reported in this paper has been deposited in the National Center for Biotechnology Information (EMBL/GenBank/DDBJ accession number
AF309870).
Disruption of CVT9 and GSA9
The chromosomal CVT9 locus was deleted by a PCR-based, one-step procedure ( strain was confirmed by tetrad analysis as described previously (
To disrupt GSA9, the S. cerevisiae ARG4 gene with its promoter was amplified by PCR using primers that contained 52 bp of 5' and 3' regions of GSA9 and 27 bp of the ARG4 gene. The PCR product was directly used to transform PPF1 cells. The resulting Arg+ clones were replica-plated to YNM plates and their inability to degrade alcohol oxidase (AOX) during glucose adaptation was determined by direct colony assay as described below. The site of ARG4 insertion within WDK09 resulting in the deletion of >90% of the GSA9 gene was verified by PCR.
Plasmid Constructions
Generation of Epitope-tagged Cvt9.
To epitope-tag Cvt9, the BamHI site in p313(CVT9) was destroyed. A new BamHI site was created after the CVT9 start codon by PCR to make p313[BHI-CVT9]. A DNA fragment encoding a 3xHA or 3xmyc epitope with BamHI sites on both sides was then ligated into the newly created BamHI site to generate the p313[3xHA-CVT9] or p313[3xmyc-CVT9] plasmid. Both plasmids complemented the prAPI accumulation phenotype of the cvt9 strain (data not shown).
The CVT9td Construct.
The method of
Constructs of CVT9 and GFPCVT9 under CUP1 Promoter Control.
The CVT9 ORF was PCR-amplified from pCVT9(414) using oligonucleotides that incorporated an in-frame XmaI site directly upstream of the start codon on the 5' primer and a SalI site after the stop codon on the 3' primer. The PCR product was digested with XmaI-SalI and subcloned into pCu416, resulting in pCuCVT9(416). The same restriction-digested PCR product was subcloned into pCuGFP(416), resulting in pCuGFPCVT9(416), which contains the CUP1-controlled GFP fused to the 5' of the CVT9 ORF.
Gsa9 was tagged with the HA epitope at the NH2 terminus by PCR amplification from genomic DNA. HA-GSA9 was inserted into the KpnI and XhoI sites of pIB2 (
Two-Hybrid Analysis
A two-hybrid screen was carried out using a truncated form of the APG1 ORF lacking the NH2-terminal eight amino acids as the bait plasmid as described (
Nitrogen Survival Assay
Nitrogen starvation analysis was done as described previously (
Alkaline Phosphatase Enzyme Assay
Induction of autophagy was estimated as activity of mutated alkaline phosphatase (ALP; Pho860p) with
-napthyl phosphate (Sigma-Aldrich) as substrate as described (
Measurements of Protein and Peroxisome Degradation
The degradation of peroxisomes in S. cerevisiae was determined by the loss of thiolase (Fox3) as described previously (
Cell Labeling, Immunoprecipitations, and Immunoblot Analysis
Radioisotopic labeling of cells, immunoprecipitations, and immunoblotting were performed as described previously (
Protease Protection and Membrane Flotation Assays
Protease sensitivity of prAPI in the cvt9td and ypt7 strains and membrane flotation experiments were performed as described previously (
Subcellular Fractionations
For subcellular fractionation experiments, cvt9 cells transformed with the pCuCVT9(416) plasmid were induced with 50 µM CuSO4 for 1 h at midlog stage and then spheroplasted as described previously (
For the biochemical characterization of Cvt9 membrane association, the total lysate was mixed with an equal volume of 1% Triton X-100 (in PS200), 6 M urea (in PS200), 0.1 M Na2CO3, pH 10.5 (in 200 mM sorbitol), or a 1.0 M salt wash (0.67 M KOAc, 0.3 M KCl). After a 5-min incubation at 25°C, the treated lysates were separated into high speed supernatant and pellet fractions by centrifugation at 100,000 g for 20 min at 4°C.
To assess prAPI binding, cvt9 spheroplasts were lysed in PS200 containing 0, 1, 2, or 5 mM MgCl2 and separated into low-speed supernatant and pellet fractions by centrifugation at 2,300 g (5,000 rpm) in an Eppendorf 5415D microcentrifuge for 5 min at 25°C. All fractionated samples were subjected to immunoblot analysis.
Optiprep Gradient Analysis
For analysis of Cvt9 by Optiprep gradients, cvt9 cells transformed with pCuCVT9(416) were incubated with 50 µM CuSO4 for 1 h to induce Cvt9 expression. At midlog stage the cells were spheroplasted and lysed in gradient buffer (PS200 containing 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and CompleteTM EDTA-free protease inhibitor cocktail). After a preclearing step at 100 g at 4°C for 5 min in an Eppendorf 5415D microcentrifuge, the lysate was separated into high-speed supernatant and pellet fractions by direct centrifugation at 100,000 g for 20 min at 4°C using a TLA100.4 rotor. The total membrane fraction was resuspended in gradient buffer and loaded to the top of a 10 ml Optiprep linear gradient (066%) and centrifuged in an SW41 rotor at 100,000 g for 16 h at 4°C. 14 fractions were collected and analyzed by immunoblotting.
Microscopy
Confocal Microscopy.
The cvt9 cells transformed with pCuGFPCVT9(416) were induced with 10 µM CuSO4 for 2 h, followed by labeling of vacuoles with FM 4-64 and viewing the cells using a confocal microscope (IRM; Leica) as described previously (
Fluorescence Microscopy.
Cells expressing GFP/HA-Gsa9 were grown in glucose or methanol for 224 h. FM 4-64 (20 µg/ml) was added and the cells were incubated for an additional 16 h. Cells grown on methanol were transferred to glucose medium for 13 h. The cells were then washed and examined immediately using an Axiophot fluorescence microscope (ZEISS). Image capture was done using a SPOT digital camera (Diagnostic Instruments, Inc.) with Adobe Photoshop® software.
Electron Microscopy.
Cells were grown for 40 h in medium supplemented with 0.5% methanol and then transferred to medium containing 2% glucose for 2 h. The cells were washed with distilled water and fixed in 1.5% KMnO4. The cells were then dehydrated, embedded in Epon 812, and sectioned for viewing on a JEOL 100CX transmission electron microscope (
Coimmunoprecipitation of Cvt9 and Apg1
Cells expressing the NH2-terminally myc-tagged Cvt9 and HA-tagged Apg1 were grown to midlog phase in YPD and spheroplasted as described previously (
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Results |
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The apg and cvt mutants share a significant genetic overlap, indicating a mechanistic convergence between autophagy and the Cvt pathway (for review see
Cvt9 Is Required for Cvt Transport but Not Autophagy
The CVT9 single-copy plasmid rescued the prAPI processing defect in the cvt9 mutant strain (Fig 1 A), although overexpression of Cvt9 using a multicopy CVT9 plasmid resulted in a moderate accumulation of prAPI. Although the CVT9 clone was isolated using the nitrogen starvation strategy, the viability of the cvt9-null strain remained relatively robust in starvation conditions when compared with a typical mutant in the autophagy pathway (e.g., cvt10/apg1; strain reflects its ability to carry out autophagy.
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The proteolytic processing of prAPI to its mature form in the vacuole provides a useful marker to assess the proper function of the Cvt pathway in nutrient-rich conditions and the autophagy pathway under starvation conditions (;
, the prAPI delivery defect observed in nutrient-rich conditions could be circumvented by a shift to nitrogen starvation conditions, suggesting that a functional autophagy pathway exists to transport prAPI to the vacuole (Fig 1 C).
To further demonstrate the competence for autophagic uptake in cvt9, we examined the recombinant cytosolic marker protein Pho8
60. Truncating the NH2-terminal transmembrane domain in the vacuolar hydrolase (ALP or Pho8) results in its cytosolic localization (
60 propeptide in the vacuole lumen generates the active form of the enzyme, which can be detected by an activity assay (Fig 1 D) or by immunoblot analysis (data not shown). Wild-type, apg1
, and cvt9
strains were transformed with an integrating plasmid that replaced the PHO8 gene with pho8
60. Cells were vegetatively grown to midlog phase and then shifted to nitrogen starvation conditions to induce autophagy. After 4 h in SD-N, vacuolar ALP activity was measured (Fig 1 D). The ALP activity in the wild-type strain increased significantly in SD-N, indicating that Pho8
60 delivery and subsequent processing to its enzymatically active form occurred in the vacuole. As expected, the shift to SD-N did not increase the ALP activity in the autophagy-defective strain apg1
. However, in cvt9
, incubation in nitrogen starvation conditions caused a strong increase in ALP activity, further demonstrating that Cvt9 is not essential for autophagic transport.
The fusion of double membrane Cvt vesicles or autophagosomes with the vacuole releases the single-membrane Cvt or autophagic body into the vacuole lumen, respectively ( strain, cells were grown in either nutrient-rich or nitrogen starvation conditions in the presence of PMSF and analyzed by electron microscopy (Fig 1 E). In nutrient-rich conditions when cells were treated with PMSF, Cvt bodies did not accumulate in the vacuole. Instead, Cvt complexes could be occasionally detected in the cytosol, confirming that the Cvt pathway is defective in the cvt9-null strain. In contrast, PMSF treatment of cvt9
cells in nitrogen starvation medium caused a substantial accumulation of autophagic bodies inside the vacuole. Taken together, these findings demonstrate that Cvt9 is required for the Cvt pathway, but does not appear to function in autophagic transport.
The Stability of prAPI Membrane Binding Is Compromised in cvt9
The delivery of prAPI via the Cvt pathway involves the assembly of prAPI oligomers into a Cvt complex that binds a pelletable membrane fraction in a salt-dependent manner ( cells were grown to midlog phase and converted into spheroplasts. The spheroplasts were lysed in an osmotic lysis buffer in the presence of titrating concentrations of MgCl2. The lysates were then separated into low-speed supernatant and pellet fractions and analyzed by immunoblots using antiserum to API as described in Materials and Methods. The apg9
strain was used as a control in which prAPI displays typical membrane-binding properties. In the absence of salt, prAPI association with the pellet fraction is severely perturbed (Fig 2 A), consistent with previous reports (
, addition of increasing concentrations of MgCl2 stabilizes the binding of prAPI to the membrane fraction; substantial prAPI binding occurred at 1 mM MgCl2 and nearly complete prAPI association with the pellet fraction was detected in 5 mM MgCl2. In contrast, in the cvt9
strain, prAPI remained largely in the supernatant fraction even at 2 mM MgCl2, with significant prAPI membrane binding occurring only at 5 mM MgCl2. These results indicate that although Cvt9 is not essential for prAPI membrane binding, the deletion of CVT9 destabilizes the interactions between prAPI oligomers and the membrane structure to which they bind.
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Cvt9 Is Required at the Stage of Cvt Vesicle Formation/Completion
The permanent loss of function in the cvt9 mutant may result in indirect, secondary phenotypes on the Cvt pathway. To determine the direct effects of Cvt9 loss of function, we generated a temperature-sensitive form of Cvt9 by constructing a chimera of Cvt9 linked to the COOH terminus of Ub-DHFR, resulting in the Cvt9td fusion protein (
To determine the step of the pathway in which Cvt9 function is required, we analyzed the state of prAPI in the cvt9td strain by protease sensitivity and membrane flotation assays. Cvt9td expression was induced at midlog phase for 1 h with 1 µM CuSO4 before spheroplasting. The cvt9td spheroplasts were radioactively labeled for 5 min followed by a nonradioactive chase reaction for 30 min at nonpermissive temperature. The labeled spheroplasts were then lysed osmotically and separated into low-speed supernatant and pellet fractions as described in Materials and Methods. The prAPI-containing pellet fractions were treated with exogenous proteinase K in the presence or absence of detergent and then immunoprecipitated with API antiserum. The supernatant and pellet fractions were also immunoprecipitated with antiserum to the cytosolic marker protein PGK to assess the osmotic spheroplast lysis efficiency. The protease sensitivity assay indicates that at nonpermissive temperature, prAPI in the cvt9td strain was accessible to exogenous proteases even in the absence of detergent treatment (Fig 2 C). The small amount of protease-resistant prAPI in the cvt9td strain probably reflects incomplete spheroplast lysis based on the appearance of a small amount of PGK in the pellet fraction. Nevertheless, the protease-protection assay of cvt9td at nonpermissive temperature suggests that Cvt vesicles had not yet formed to enclose prAPI in this strain. The ypt7 mutant was used as the control strain. Ypt7 is a rab guanosine triphosphatase required for Cvt vesicle fusion with the vacuole, and thus the ypt7
strain accumulates prAPI in a protease-protected state (Fig 2 C;
In the membrane flotation gradient analysis, membrane-associated proteins in a cell lysate migrate to the top of a step gradient and are collected as the "float" (F) fraction. Soluble proteins remain in the nonfloat (NF) fraction and proteins associated with large protein complexes are recovered in the gradient pellet fraction (P2). The radiolabeled prAPI pellet fraction derived from the lysate of the cvt9td strain at nonpermissive temperature was loaded on the bottom of a step gradient and subjected to centrifugation. The majority of prAPI from the cvt9td strain was recovered in the float fraction, indicating that prAPI associates with a floatable membrane source (Fig 2 D). As a positive control for the flotation gradient analysis, we examined the prAPI pellet fraction derived from a temperature-conditional allele of apg9 (apg9ts). Consistent with our recent study on Apg9, prAPI from the apg9ts strain associates with a membrane source and is recovered in the float fraction (
Cvt9 Is an Oligomeric, Coiled Coil, Peripheral Membrane Protein
To further understand Cvt9 function in the Cvt pathway, we next characterized the biosynthesis of the Cvt9 protein. Polyclonal antiserum raised against synthetic Cvt9 peptides recognized a 135-kD band by immunoblot analysis, in agreement with its predicted molecular mass (data not shown). Detection of Cvt9 increased in cvt9 cells transformed with the CVT9 multicopy plasmid and was absent in the cvt9
cells, suggesting that the antiserum is specific for the Cvt9 protein. Analysis of the Cvt9 amino acid sequence reveals a significant coiled coil secondary structure between amino acid residues 542 and 851 (http://nightingale.lcs.mit.edu/cgi-bin/score). The P. pastoris homologue, Gsa9, also contains a predicted coiled coil secondary structure motif between amino acids 811 and 1027. Because a common feature of proteins containing a coiled coil domain is the ability to form higher-ordered quaternary structures, we investigated the possibility that Cvt9 forms oligomers. Extracts from cells expressing NH2-terminally myc-tagged Cvt9 and HA-tagged Cvt9 were prepared and the immunocomplex containing HA-Cvt9 was immunoprecipitated under native, nonreducing conditions with antiserum to the HA epitope. A subsequent anti-myc epitope immunoblot detected the myc-tagged Cvt9 from the original immunoprecipitated HACvt9 immunocomplex, indicating that myc-Cvt9 associates with HACvt9 (Fig 3 A). These findings indicate that Cvt9 forms homodimers or potentially larger oligomeric structures.
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The subcellular location of Cvt9 was initially examined by subcellular fractionation and differential centrifugation procedures. The cvt9 strain was transformed with a plasmid containing CVT9 behind the CUP1 copper regulable promoter (
strain was completely reversed at this level of Cvt9 expression (data not shown). The spheroplasts were lysed osmotically and separated into low-speed supernatant and pellet fractions (S13 and P13, respectively). The S13 supernatant fraction was further resolved into high-speed supernatant and pellet fractions (S100 and P100, respectively). The majority of Cvt9 fractionated to both the P13 and P100 fractions, whereas only a minor population appeared in the high-speed supernatant fraction (Fig 3 B). We next investigated the nature of the Cvt9 pellet association (Fig 3 C). The spheroplasts were lysed osmotically and treated with lysis buffer, Triton X-100, urea, Na2CO3, or a high salt wash as described in Materials and Methods. The treated lysates were then subjected to high-speed centrifugation and separated into total membrane and supernatant fractions. The buffer-only treatment confirmed that the majority of Cvt9 remained bound to a membrane pellet fraction. However, treatment with urea, Na2CO3, and the high-salt wash, conditions that remove peripheral membrane proteins, all stripped a substantial portion of Cvt9 from the membrane pellet into the supernatant fraction. In addition, examination of Cvt9 by membrane flotation gradient analysis indicated that Cvt9 was recovered in the "float" fraction, suggesting that it associated with a floatable membrane (data not shown). Therefore, we propose that Cvt9 is a peripheral membrane protein (Fig 3 C). In contrast, the integral membrane protein Dpm1 remained in the pellet fraction under all conditions, except when membranes were solubilized with detergent. Interestingly, Cvt9 remained in the pellet fraction after treatment with Triton X-100, suggesting that Cvt9 multimers may form a larger detergent-resistant protein complex.
Cvt9 Localizes to a Distinct Perivacuolar Compartment
To investigate the identity of the membrane compartment with which Cvt9 associates, a GFP fusion to the NH2 terminus of Cvt9 was examined. Under the control of the CUP1 promoter, GFPCvt9 expression complemented the prAPI defect in the cvt9 strain, indicating that it was a functional chimera (data not shown). At midlog stage, cvt9
cells expressing GFPCvt9 were labeled with FM 4-64 to mark the vacuoles and examined by confocal microscopy. GFPCvt9 appeared to be concentrated at prominent punctate structures directly adjacent to FM 4-64labeled vacuoles (Fig 4 A). A weak, cytosolic distribution of GFPCvt9 was also detected, consistent with the ratio of Cvt9 found in the membrane pellet and cytosolic supernatant fractions by biochemical fractionation analysis (compare Fig 3 B and 4 A).
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The confocal micrographs of GFPCvt9 suggested residence in a compartment proximal to the vacuole or directly on the vacuolar membrane in the form of a concentrated patch. To identify the Cvt9 compartment relative to known membrane marker proteins, we next examined Cvt9 subcellular localization by linear Optiprep density gradients (Fig 4B and Fig C). Expression of Cvt9 was induced with 50 µM CuSO4 for 1 h before spheroplasting. A total membrane fraction was prepared from osmotically lysed spheroplasts and resolved on Optiprep gradients as described in Materials and Methods. Immunoblot analysis of the collected gradient fractions indicated a peak for the Pho8-localized vacuole compartment in fraction 1, whereas the Cvt9 compartment migrated to a much denser region, with a peak in fraction 10 (Fig 4 B). This finding demonstrates that the perivacuolar Cvt9 compartment is distinct from the vacuole. Furthermore, the Cvt9 compartment did not appear to cofractionate with any other resident organelle marker proteins, including Dpm1 (ER), Kex2 (late Golgi apparatus), and Pep12 (endosome) (Fig 4 C). Taken together, the identity of the perivacuolar compartment to which Cvt9 localizes appears to be distinct from known organelles of the endomembrane system.
Cvt9 Physically Interacts with the Apg1 Kinase
Cvt9 is present in a detergent-resistant form, presumably a protein complex, which localizes to a distinct membrane compartment (Fig 3 and Fig 4). We next identified an additional factor of the Cvt9 protein complex. A two-hybrid analysis to identify proteins that associate with the autophagy component Apg1 identified Cvt9 as a potential interacting partner ( strain was transformed with the plasmid encoding the myc-tagged Cvt9 fusion protein with or without a second plasmid encoding Apg1. Cell extracts were immunoprecipitated with antiserum to Apg1 under native, nondenaturing conditions as described in Materials and Methods. The precipitated immunocomplexes were then subjected to a denaturing SDS-PAGE/immunoblot procedure with antiserum to the myc epitope. In cells expressing both mycCvt9 and Apg1, the immunocomplexes precipitated with the Apg1 antiserum also isolated the myc-tagged Cvt9 protein, thus confirming the two-hybrid data that Apg1 and Cvt9 physically interact (Fig 5, lane 5).
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Cvt9 and Its P. pastoris Homologue, Gsa9, Are both Required for Peroxisome Degradation
We identified GSA9 as an essential component for the glucose-stimulated peroxisome degradation pathway in P. pastoris. Sequence analysis revealed GSA9 to be a structural and functional homologue of CVT9. Both proteins contain a predicted coiled coil domain. However, although many proteins encoded by the S. cerevisiae genome share common residues in the coiled coil domain, only Cvt9 shows extensive homology with its P. pastoris counterpart over the entire sequence. More specifically, a BLAST search with the NH2-terminal and COOH-terminal Gsa9 domains (amino acids 1810 and 10281316, respectively) flanking its predicted coiled coil region was performed against all S. cerevisiae proteins (http://genome-www2.stanford.edu/ cgi-bin/SGD/nph-blast2sgd). The BLAST results identified the corresponding NH2-terminal and COOH-terminal domains flanking the coiled coil region of Cvt9 as the only sequences with statistically significant similarity to Gsa9 (P value, 3.9 x 10-19 and 3.9 x 10-17, respectively). This sequence analysis indicates that Cvt9 is the sole homologue of Gsa9 in S. cerevisiae and does not represent one member in a family of related proteins.
To determine if Cvt9 was also required for the specific degradation of peroxisomes in S. cerevisiae, we examined the degradation of Fox3, the peroxisomal thiolase enzyme, following a previously described method for examining pexophagy in S. cerevisiae ( and wild-type cells by growth in oleic acid medium. Upon shift to SD-N medium to induce pexophagy, peroxisome degradation was examined by monitoring cellular Fox3 levels over the indicated time course (Fig 6 A). In wild-type cells, Fox3 levels decreased significantly over the time course, whereas they remained constant in cvt9
cells. This finding suggests that Cvt9 is required for the specific, vacuolar delivery of not only prAPI via the Cvt pathway, but also peroxisomes via the pexophagy pathway in S. cerevisiae.
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The P. pastoris gsa9 mutant was further characterized to better understand the cellular function of Gsa9. Peroxisomes and the expression of resident enzymes, such as AOX, were induced in methanol-containing medium. The peroxisomes were then degraded upon shift to glucose-containing medium. After a 6-h incubation in glucose adaptation conditions, AOX activity was measured (Fig 6 B). Wild-type GS115 cells displayed significant loss of AOX activity, indicating that excess peroxisomes were degraded via pexophagy. As a negative control, gsa7 cells were analyzed. GSA7 is the P. pastoris homologue of APG7, an essential component for pexophagy as well as the Cvt pathway and autophagy (
As we have noted, Cvt9 does not play an integral role in starvation-induced autophagy (Fig 1). If Gsa9 functions in an analogous manner, then gsa9 mutants should also be competent for autophagic degradation. Autophagy accounts for the majority of the cellular degradative capacity during starvation conditions (for review see
The process of micropexophagy can be categorized into distinct stages ( strains by electron microscopy (Fig 6 D). In wild-type cells, peroxisomes could be occasionally detected undergoing degradation in the vacuolar lumen. However, both gsa9-2 and gsa9
cells exhibited a micropexophagy defect at a middle/late stage in which vacuolar extensions only partially surround the peroxisome but do not complete the engulfment process.
Gsa9 Is Recruited to the Vacuolar Membrane during Peroxisome Uptake
To further characterize the role of Gsa9 in micropexophagy, we examined its localization by fluorescence microscopy using a functional GFP/HA-Gsa9 fusion protein. This construct was able to complement the gsa9 phenotype (data not shown). In cells that have not been subjected to glucose adaptation conditions, GFP/HA-Gsa9 appeared localized to one or two structures adjacent to the vacuole (data not shown). In addition, a weak GFP/HA-Gsa9 signal was detected coincident with the FM 4-64labeled vacuoles. The punctate pattern of GFP/HA-Gsa9 appeared nearly identical to the localization pattern of GFPCvt9, although we could not detect GFPCvt9 on the vacuole (Fig 4 A). When cells expressing GFP/HA-Gsa9 were grown in methanol medium to proliferate peroxisomes and then subjected to adaptation in glucose medium, the GFP/HA-Gsa9 signal localized predominantly to the vacuolar membrane (Fig 7, top). To compare GFP/HA-Gsa9 localization to that of peroxisomes, we constructed a blue fluorescent protein (BFP) tagged with the serine-lysine-leucine COOH-terminal peroxisomal targeting signal (BFP-SKL; Fig 7, middle). In glucose adaptation conditions, the intensity of GFP/HA-Gsa9 appeared strongest at the vacuolar regions that surrounded the peroxisomes during the engulfment process (Fig 7, bottom). GFP/HA-Gsa9 also formed punctate structures that appeared continuous with its vacuolar membrane labeling pattern. The distribution of GFP/HA-Gsa9 before and after glucose adaptation suggests that Gsa9 localizes to regions of the vacuolar membrane that contact peroxisomes during micropexophagy.
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Discussion |
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Autophagy and the Cvt pathway are overlapping processes that share most components. However, the Cvt pathway operates in a biosynthetic capacity during vegetative conditions, whereas autophagy is primarily a degradative process that is induced by starvation. Both pathways involve dynamic membrane rearrangements to deliver cytosolic contents to the vacuole. Morphological studies suggest that the two pathways normally operate exclusively of one another. The regulatory mechanisms and structural differences that control the conversion between the Cvt pathway and autophagy are not well understood. To gain further insight into the factors that are distinct between these pathways, we have examined a protein component, Cvt9, and its P. pastoris homologue, Gsa9, which appear to be specific to the Cvt pathway and micropexophagy, respectively.
Cvt9 Forms a Protein Complex Comprised of Cvt/Apg Phosphoproteins
Cvt9 exists primarily as a peripherally bound membrane protein (Fig 3B and Fig C). Coimmunoprecipitation results demonstrate that it forms homodimers or even higher order homooligomers (Fig 3 A), potentially through a coiled coil assembly mechanism, as indicated by secondary structural analysis. Consistent with our recent two-hybrid results (
The differential effects that Cvt9, Apg1, Apg13, Apg17, and Vac8 exert on the Cvt and autophagy pathways suggest that these components may serve to regulate the conversion between Cvt transport and autophagy. Apg1, Apg13, Cvt9, and Vac8 are all phosphoproteins, and the phosphorylation state and affinity of Apg13 to Apg1 are regulated by nutrient conditions through the Tor kinase cascade (
The Cvt9 Protein Complex Localizes to a Novel Perivacuolar Compartment
The subcellular distribution of GFPCvt9 supports an increasingly familiar localization pattern among Cvt and autophagy components (
Mechanistic Similarities between the Cvt/Autophagy Pathways and Micropexophagy
In P. pastoris, the specific degradation of excess peroxisomes during glucose adaptation entails the sequestration of peroxisomes by the vacuole in the process of micropexophagy. The mechanistic details of how a Cvt vesicle or an autophagosome forms to sequester cargo appear morphologically similar to the vacuole-mediated engulfment of peroxisomes. In each case, the sequestration of cargo by a membrane source results in the formation of a double-membrane structure. In the case of micropexophagy, the enwrapping membrane is the vacuole itself whereas the membrane source in the formation of Cvt vesicles and autophagosomes remains to be determined.
We have identified Gsa9 as the P. pastoris homologue of Cvt9. The functional analysis of Gsa9 corroborates and may provide new insights into the role of Cvt9 in S. cerevisiae. Neither Gsa9 nor Cvt9 is required for autophagy, whereas both are essential for peroxisome degradation (Fig 1 and Fig 6). The degradation of peroxisomes in S. cerevisiae may occur by micropexophagy (
The prAPI phenotype in cvt9 supports a similar defect in the Cvt pathway. To characterize the direct effect of Cvt9 inactivation, we constructed a Cvt9 temperature-degron fusion protein (Cvt9td), a novel temperature-conditional form of Cvt9 (Fig 2 B). At nonpermissive temperature, prAPI in the cvt9td strain remained protease accessible (Fig 2 C). In addition, prAPI was able to float through a step gradient in a detergent-dependent manner, suggesting that it associated with a floatable membrane (Fig 2 D). Therefore, the cvt9 defect in the Cvt pathway may be at a middle-to-late stage in Cvt vesicle formation, analogous to the gsa9 mutant phenotype for micropexophagy.
Localization of Gsa9 Versus Cvt9
A key difference between Gsa9 and Cvt9 is their subcellular localization patterns. Before the induction of micropexophagy, Gsa9 appears to be localized to both a punctate structure in the perivacuolar region and diffusely distributed on the vacuole (data not shown). However, upon glucose adaptation, a population of Gsa9 strongly localizes to regions of the vacuolar membrane that are in contact with peroxisomes in the process of being sequestered into the vacuole (Fig 7). In contrast, the localization of Cvt9 to the perivacuolar compartment appears constant regardless of nutrient status. If Cvt9 functions analogously to Gsa9 in the Cvt pathway of S. cerevisiae, then it may directly participate in the enwrapping of prAPI-containing Cvt complexes to form the double-membrane Cvt vesicles. Furthermore, if Cvt9 acts at the site of cargo sequestration in an analogous manner to the function of Gsa9 at the vacuole during peroxisome engulfment, then the Cvt9 localization pattern would identify the perivacuolar compartment as the donor membrane source for Cvt vesicles. Additional studies will be required to test this assertion.
Potential Function of Gsa9 and Cvt9
Given that the mechanisms of the Cvt pathway and pexophagy are quite similar to autophagy (for review see strain (Fig 1 C). If a deletion in CVT9 did not affect the selective nature of prAPI import, then one would predict that the entire population of prAPI would enter the vacuole via autophagy. The near complete reversal of prAPI under starvation conditions is observed in some API propeptide mutants that retain the ability to bind to the membrane (
60 via autophagy plateaus at
30%. Therefore, the loss of cargo selectivity in the cvt9
strain may result in the recognition of prAPI as another nonspecific molecule during autophagy. Further analysis of Cvt9 and the proteins that associate with it will provide additional information about the conversion between the Cvt pathway and autophagy as well as the mechanism that modulates specificity in the Cvt pathway and pexophagy.
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Footnotes |
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1 Abbreviations used in this paper: ALP, alkaline phosphatase; AOX, alcohol oxidase; API, aminopeptidase I; BFP, blue fluorescent protein; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent protein; HA, hemagglutinin; PGK, phosphoglycerate kinase; prAPI, precursor API; SMD, synthetic minimal medium; td, temperature degron; YNB, yeast nitrogen base.
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Acknowledgements |
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We thank Maria Hutchins for advice and helpful discussions on pexophagy; Kimberley Eggerton, Andrew Bevan, and Denny Player for technical assistance; and Wei-Pang Huang, Chao-Wen Wang, and Drs. Sarah Teter and Hagai Abeliovich for critical reading of the manuscript. We also thank Dr. Jürgen Dohmen for supplying the plasmids used in constructing the cvt9td mutant and Dr. Dennis Thiele for supplying the vectors containing the yeast CUP1 inducible copper promoter.
This work was supported by Public Health Service grant GM53396 from the National Institutes of Health to D.J. Klionsky, Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan to Y. Ohsumi, a grant from The Norwegian Cancer Society to P.E. Stromhaug, and National Science Foundation grant MCB-9817002 to W.A. Dunn, Jr.
Submitted: 23 October 2000
Revised: 30 January 2001
Accepted: 20 February 2001
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