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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Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.
Key Words: autophagy, lysosome, protein targeting, vacuole, yeast
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Introduction |
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Stress conditions elicit an array of specialized responses in eukaryotic cells. To survive nutritional deprivation, cells must quickly mobilize the transport of nonessential cytoplasmic materials to the principal degradatory organelle, the vacuole/lysosome, for subsequent digestion and reuse in essential biosynthetic processes (reviewed in
The starvation-induced autophagy pathway provides the molecular machinery to sequester bulk cytoplasm destined for vacuolar degradation (
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Analysis of the autophagy and cvt mutants indicates that many of the characterized components are required at an early stage(s) in vesicle formation. Mutants defective in this part of the pathway all possess a phenotype in which prAPI binds to a pelletable membrane but remains accessible to exogenous protease treatment, indicating that a completed vesicle has not yet formed (
Autophagosomes are substantially larger than Cvt vesicles that form under vegetative conditions (
The recruitment of soluble vesicle coat factors to target membranes is a critical step in vesicle formation (
In this study, we examined the factors required for the recruitment of the soluble Aut7 protein to the membrane, an early step leading to Cvt and autophagic vesicle formation. After synthesis in the cytosol, Aut7p is proteolytically processed in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the Apg conjugation complex. In this study, we assign a functional role for Aut1p and the Apg conjugation system as mediators of Aut7p membrane recruitment.
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Materials and Methods |
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Yeast Strains
The yeast strains used in this study are listed in Table 1.
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Antibodies
To prepare antiserum to Aut1p, synthetic peptides corresponding to amino acids 138156 and 245263 of Aut1p were synthesized with a Cys residue at the amino terminus and conjugated individually to keyhole limpet hemocyanin (Multiple Peptide Systems). Standard procedures were used to generate antiserum in a New Zealand white rabbit (
Materials and Growth Media
The lipophilic dye FM 4-64 was from Molecular Probes. Ficoll-400 was from Amersham Pharmacia Biotech. Immobilon-P (polyvinylidene fluoride) was from Millipore. CompleteTM EDTA-free protease inhibitor cocktail was from Roche Molecular Biochemicals. Oligonucleotides were synthesized by Operon. Oxalyticase was from Enzogenetics. YNB was from DifCo. Copper-free YNB was from Bio101. All restriction enzymes and VentR DNA polymerase were from New England Biolabs, Inc. The pGFP(416)SK and pGFP(426)SK vectors for expressing green fluorescent protein (GFP) fusions were a gift from Dr. Tom Vida (University of Texas Health Sciences Center, Houston, TX). The copper-inducible promoter-based vectors, pCu416, pCu414, and pCu426, were gifts from Dr. Dennis J. Thiele (University of Michigan, Ann Arbor, MI;
Yeast strains were grown in synthetic minimal medium (SMD; 0.67% yeast nitrogen base, 2% glucose and auxotrophic amino acids and vitamins as needed). Synthetic minimal medium containing 2% glucose without ammonium sulfate or amino acids (SD-N) was used for nitrogen starvation experiments.
Disruption of the AUT1, AUT2, and AUT7 Chromosomal Loci
The chromosomal AUT1 locus was deleted by a PCR-based, one-step procedure (
Construction of Plasmids
To construct the pAUT1(426) plasmid, the AUT1 ORF and flanking sequences were PCR-amplified from a wild-type yeast genomic DNA preparation. The 5' and 3' oligonucleotides used for the amplification were 5'-CAGTGTGCCCATTTTTTACTCGAGTGTCGCCGAATTCGTTGGTAGAGAACC-3' and 5'-GAAAGTGGTTTTAAGGAAGAAGCTTGTTCCTGCATATTTATAGTTAATTCC-3', respectively. The 5' oligonucleotide contains a XhoI site and the 3' oligonucleotide contains a HindIII site. The AUT1 PCR product was subcloned into pRS426 using the XhoI and HindIII sites. The resulting pAUT1(426) clone contains the AUT1 ORF with 175 bp of upstream and 98 bp of downstream sequences.
The AUT2 clone was also PCR-amplified from a wild-type yeast genomic DNA preparation. The 5' and 3' oligonucleotides used for the amplification were 5'- GCTACCACGTGCATGCCTACGTAAGCTTCCTTGATAATGACTATGCC-3' and 5'-GCGATGACTAGGCATTTTGGACTTCGACTCGAGTTTATACCACCGTTGTCTTAATCTTC-3', respectively. The 5' oligonucleotide contains a HindIII site and the 3' oligonucleotide contains a XhoI site. The AUT2 PCR product was subcloned into pRS416 using the HindIII and XhoI sites. The resulting pAUT2(416) clone contains the AUT2 ORF with 269 bp of upstream and 227 bp of downstream sequences.
To make the AUT2GFP fusion construct, the AUT2 ORF was PCR-amplified using the pAUT2(416) construct as the template. The 5' and 3' oligonucleotides used for the amplification were 5'-GCTTCCTTGATAATGAGCTCGCCTTTTTGCC-3' and 5'-GAATTCCTTACACTAGACTAGTTCATCAATAGG-3', respectively. The 5' oligonucleotide contains a SacI site and the 3' oligonucleotide contains a SpeI site that starts 7 bp upstream of the stop codon. The PCR product was subcloned into the pGFP(416)SK and pGFP(426)SK vectors using the SacI and SpeI sites. The resulting pAUT2GFP(416) and pAUT2GFP(426) constructs contain 253 bp of upstream AUT2 sequences followed by the AUT2 ORF, and then the inframe GFP ORF and termination sequences.
A PCR-based approach was used to create a strain expressing an Aut2HAp fusion protein from the AUT2 chromosomal locus. The 5' oligonucleotide encoded 40 bp of the COOH-terminal AUT2 ORF followed by the start of the triple HA sequences; the 3' oligonucleotide contained 40 bp of 3' untranslated region of AUT2 followed by ME3 vector sequences adjacent to the HIS5 marker: 5'-GGAAACGGTAGGTATTCACAGTCCTATTGATGAAAAATGCTACCCATACGATGTTCCT-3' and 5'-GGATGATCAAACGAATAAGAACAAAAGAACTGGATACCTCGTCGACGGTATCGATAAG-3'. The ME3 vector was used as the PCR template. The resulting PCR product consisted of 40 bp of the COOH-terminal AUT2 ORF, followed by the triple HA epitope sequences, the termination cassette, the HIS5 auxotrophic marker, and, finally, 40 bp of the AUT2 3' untranslated region. The PCR product was transformed into the SEY6210 wild-type strain. Chromosomal integration of the PCR product by homologous recombination resulted in an inframe fusion of the triple-HA epitope sequence to the COOH-terminal coding sequence of the AUT2 ORF. Recombinants were selected on minimal plates lacking histidine. A strain expressing the Aut2HAp fusion protein was confirmed by PCR and by antiHA immunoblots.
The construct for the fusion protein of Aut7p followed by GFP at its COOH terminus [pAUT7GFP(416)] was made by PCR amplifying the AUT7 ORF and upstream sequences using the pAUT7(424) plasmid (
To make NH2-terminal GFP fusion proteins under the control of a regulable promoter, the CUP1 promoter cassette from the pCu(416) vector was subcloned into pGFP(416)SK by a SacISpeI digest, thus creating the pCuGFP(416) vector. The CUP1-promoter driven construct for the fusion of GFP to the NH2 terminus of Aut7p was then made by PCR amplifying the AUT7 ORF using pAUT7(416) as the template. The 5' and 3' oligonucleotides used for the PCR amplification were 5'-CTAGAGACCCGGGGTCTACATTTAAGTCTGAATATCC-3' and 5'-CGCTTCATTTCTTTTCATACTCGAGACTACCTGCCAAATG-3', respectively. The 5' oligonucleotide contains an XmaI site that is in the same reading frame as the end of the GFP ORF of pCuGFP(416) and the 3' oligonucleotide contains an XhoI site. The PCR product was subcloned into pCuGFP(416) by an XmaIXhoI digest, resulting in the pCuGFPAUT7(416) plasmid.
Subcellular Fractionation
For subcellular fractionation experiments, strains were grown to midlog phase in SMD medium and spheroplasted as described earlier (
Membrane Flotation and Protease Sensitivity Analyses
The procedures for the membrane flotation and protease sensitivity were performed following a previously described protocol (
Confocal and Conventional Fluorescence Microscopy
For confocal microscopy analysis of yeast strains expressing the GFPAut7p fusion protein under the control of the CUP1 copper-inducible promoter, cells were grown to midlog phase in SMD medium lacking copper and uracil. GFPAut7p fusion protein expression was induced with 10 or 50 µM CuSO4 for 23 h, followed by FM 4-64 labeling at a final concentration of 10 µg/ml for 20 min. The FM 4-64labeled cells were incubated in YPD for 90 min to allow uptake of the dye, and then viewed directly or shifted to SD-N medium for 3 h to induce autophagy. Cells were examined on a Leica DM IRB confocal microscope. Eight scans of each optical section were averaged for the final images, except in Fig 2B and Fig C (below), where single scans of each image were taken.
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Results |
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Aut7p Membrane Binding Requires an Aut2p-dependent Proteolytic Cleavage Event
Aut7p is the first component to be identified on Cvt vesicles and autophagosomes and, as such, serves as a valuable vesicle marker for the pathway (
Concomitant with the confocal microscopy analysis of the Aut7pGFP fusion protein, we wanted to confirm that the fusion protein was expressed properly (Fig 1 C). Yeast strains were transformed with the pAUT7GFP plasmid and grown under vegetative conditions to midlog phase in SMD medium. Protein extracts were prepared from each strain and subjected to immunoblot analysis with antiAut7p and GFP antibodies. In both wild-type and aut7 cells transformed with the pAUT7GFP plasmid, a proteolytic processing event cleaves the Aut7pGFP fusion into separate Aut7p and GFP proteins (Fig 1 C, 3 and 4). The Aut7p moiety resulting from cleavage appeared identical in electrophoretic mobility to the Aut7p expressed from the pAUT7 plasmid (Fig 1 C, compare 2 with 3), suggesting that the cleavage occurred near the COOH terminus of the protein. Therefore, proteolytic cleavage of the Aut7pGFP fusion releases the GFP domain into the cytosol, consistent with the confocal microscopy observations in Fig 1 A, while the Aut7p portion of the fusion protein functions to complement the prAPI transport defect of the aut7
strain.
We next wanted to identify components that could mediate the proteolytic processing of Aut7p. One potential candidate was Aut2p, which has been demonstrated to interact with Aut7p by affinity chromatography ( and apg9
, Aut7p processing still occurred. These findings indicate that Aut2p mediates the COOH-terminal proteolytic cleavage of Aut7p. Because these strains were grown in rich medium, high expression of the endogenous Aut7p was not induced. As a result, the only detectable Aut7p in our experimental conditions was derived from the pAUT7 and pAUT7GFP plasmids. Taken together, the Aut2p-mediated cleavage of the Aut7pGFP fusion protein agrees with the recent study by
Aut7p is a hydrophilic, soluble protein that is recruited to forming autophagosomes. To determine whether the COOH-terminal processing of Aut7p was required for its membrane association, subcellular fractionations were performed. Aut7p in the aut2 strain exists in the uncleaved form, while in other apg mutants, such as apg9
, Aut7p exists in the proteolytically modified state (Fig 1 C). Therefore, cells from aut2
and apg9
strains were grown to midlog phase under vegetative conditions, shifted to nitrogen starvation conditions for 2 h to induce autophagy and Aut7p expression, and then converted to spheroplasts. The spheroplasts were then subjected to differential osmotic lysis conditions that allow lysis of the plasma membrane while retaining the integrity of subcellular compartments. The lysed spheroplasts were then centrifuged to separate the lysates into low-speed supernatant and pellet fractions (S13 and P13, respectively). The S13 fraction was then further separated into high-speed supernatant and pellet fractions (S100 and P100, respectively). In the apg9
strain, where Aut7p has undergone the proteolytic cleavage event, a significant population of Aut7p appeared in the low-speed pellet fraction (Fig 1 D). In contrast, the uncleaved Aut7p in the aut2
strain was distributed entirely in the soluble supernatant fractions. Taken together, these results indicate that the Aut2p-mediated COOH-terminal processing of Aut7p is required for Aut7p membrane association, in agreement with recent findings by
A GFP Fusion to the NH2 Terminus of Aut7p is an In Vivo Marker for both the Cvt Pathway and Autophagy
Cleavage of GFP from the COOH terminus of Aut7p provided a useful diagnostic tool for examining the novel proteolytic processing reaction that modifies the Aut7 protein. However, the Aut7pGFP construct could not be used to follow localization of Aut7p. We next explored whether a GFP fusion to the NH2 terminus of Aut7p would provide an intact, functional, fusion protein that could be used to monitor the Cvt and autophagy pathways in vivo. The NH2-terminal GFPAut7p fusion was placed under the control of a copper-inducible promoter (pCuGFPAUT7) and transformed into aut7 cells. Expression of the GFP Aut7p fusion protein was induced by the addition of 50 µM CuSO4 for 3 h before immunoblot analysis. The expressed GFPAut7p fusion protein was maintained in its full-length form and complemented the prAPI accumulation phenotype in the aut7
strain, indicating that it was an intact, functional fusion protein (Fig 2 A).
Previous studies have demonstrated that Aut7p is localized to both Cvt vesicles and autophagosomes that are targeted to the vacuole () accumulate these Aut7p-containing, single-membrane vesicles inside the vacuole. If the GFPAut7p fusion protein follows the itinerary of Cvt vesicles and autophagosomes, then it would also accumulate inside Cvt and autophagic bodies in a pep4
strain under nutrient-rich and starvation conditions, respectively. To test this hypothesis, pep4
cells transformed with the pCuGFPAUT7 plasmid were grown in nutrient rich conditions to detect Cvt bodies or in nitrogen starvation conditions (SD-N) to observe autophagic bodies inside the vacuole (Fig 2, BD).
To examine Cvt bodies, fusion protein expression was induced with a relatively low concentration of CuSO4 (10 µM) for 3 h to reflect the lower level of Aut7p expression under nutrient-rich conditions. After labeling the vacuoles with FM 4-64, the cells were examined by confocal microscopy. Under these nutrient-rich conditions, the GFPAut7p fusion protein was localized to multiple Cvt bodies inside the FM 4-64labeled vacuoles (Fig 2 B). To demonstrate the dynamic nature of these accumulated Cvt bodies inside the vacuole, we performed a time-series analysis of a single focal plane of pep4 cells expressing GFPAut7p under nutrient-rich conditions (Fig 2 C). The sequential video frames, taken every 4.4 s, show GFPAut7p-labeled Cvt bodies inside FM 4-64labeled vacuoles moving in and out of the focal plane of the optical section. These findings demonstrate that GFPAut7p, like the endogenous Aut7 protein, is recruited to Cvt vesicles that are transported to the vacuole via the Cvt pathway.
To view autophagic bodies, GFPAut7p expression was induced with 50 µM of CuSO4 for 3 h and shifted to SD-N for 3 h to induce autophagy. Under these conditions, the FM 4-64labeled vacuoles were full of GFPAut7p-labeled autophagic bodies (Fig 2 D). Taken together, these observations confirm that the GFPAut7p fusion protein provides a useful in vivo marker for both the Cvt pathway and autophagy. Consistent with previous studies, the autophagic bodies were much larger than the Cvt bodies, and allowed for easier detection by confocal microscopy (compare Fig 2B with D). Furthermore, because autophagy conditions induce the expression of Aut7p, thus facilitating its immunodetection, all subsequent Aut7p experiments were performed under autophagy conditions.
The Aut2p-mediated proteolytic processing of Aut7p is a requisite step in its subsequent binding to the P13 membrane fraction (Fig 1 D). We next characterized the nature of the P13 membrane fraction to which Aut7p binds by examining the GFPAut7p marker by confocal microscopy. The apg1 and aut2
strains were transformed with the pCuGFPAUT7 plasmid and grown to midlog phase in SMD. GFPAut7p expression was induced with 50 µM CuSO4 for 3 h. After labeling vacuoles with FM 4-64, the cells were shifted to SD-N medium to induce autophagy. In the apg1
strain, where Aut7p processing occurs (Fig 1 C), GFPAut7p localized to prominent punctate structures and was also seen in a diffuse cytosolic population (Fig 3). These punctate structures were always detected in a perivacuolar region and generally numbered from one to several structures per cell. With a few notable exceptions (discussed below), a population of GFPAut7p displaying a punctate pattern was found in all of the apg mutants examined (data not shown). In contrast, GFPAut7p in the aut2
strain, where Aut7p processing did not occur, remained entirely diffuse in the cytosol (Fig 3). This result supports the fractionation data (Fig 1 D), as well as the findings of
The physical interaction between Aut2p and Aut7p suggested that the localization of Aut2p might display the same membrane-binding pattern observed for Aut7p. To examine the subcellular localization of Aut2p, a fusion construct of AUT2 followed by a triple HA epitope tag was integrated at the AUT2 locus. The Aut2HAp integration was functional, as prAPI maturation in the aut2::AUT2HA strain appeared similar to the wild-type strain (Fig 4 A). To determine the subcellular fractionation pattern of Aut2HAp, cells expressing Aut2HAp were grown to midlog stage in SMD, and then shifted to SD-N medium for 2 h before spheroplasting. The spheroplasts were then lysed in differential osmotic lysis buffer and separated into S13 and P13 fractions. The S13 fraction was further resolved into S100 and P100 fractions. Immunoblot analysis revealed Aut2HAp to be entirely in the supernatant fractions, along with the PGK cytosolic marker (Fig 4 B). The cytosolic fractionation pattern of Aut2HAp was also observed in cells grown under vegetative conditions (data not shown). The Aut2HAp cytosolic fractionation pattern was also supported by an Aut2pGFP fusion protein, which complemented the prAPI defect in the aut2 strain (Fig 4 A), and exhibited a diffuse cytosolic localization pattern (data not shown). Further, unlike Aut7p, Aut2p expression was not induced in starvation conditions, suggesting that it may act in a catalytic manner (data not shown). Taken together, the findings above indicate that the soluble autophagy component, Aut2p, acts to mediate the cleavage of Aut7p in the cytosol. After proteolytic processing, Aut7p binds to a punctate, perivacuolar, membrane structure that can be fractionated into a low-speed pellet by centrifugation.
Aut1p Function Is Required for Aut7p Recruitment to the Membrane
Comprehensive two-hybrid analyses of the yeast genome have recently elucidated a number of novel interactions among autophagy components (see Fig 9 A; strain demonstrated both defects in prAPI transport as well as starvation-induced transport of bulk cytoplasm to the vacuole (
strain was grown to midlog phase and shifted to SD-N medium to induce autophagy and Aut7p expression. Subcellular fractionations and centrifugations separated the lysate into S13, P13, S100, and P100 fractions. Immunoblot analysis with antiserum to Aut7p indicated that Aut7p remained entirely in a cytosolic fraction (Fig 5 A). These observations were also supported by examination of the GFPAut7p fusion protein in the aut1
background (Fig 5 B). The aut1
strain transformed with pCuGFPAUT7 was induced with 50 µM CuSO4 at midlog phase for 3 h, labeled with FM 4-64 to mark the vacuoles, and shifted to SD-N medium for 3 h before examination by confocal microscopy. The GFPAut7p fusion protein was distributed diffusely in the cytoplasm (Fig 5 B).
To better understand the role of Aut1p in Aut7p function, we also examined the subcellular location of Aut1p (Fig 5 C). Wild-type cells were grown to midlog phase, and then shifted to SD-N medium for 2 h before converting them into spheroplasts. Differential osmotic lysis and centrifugation steps separated the lysate into S13, P13, S100, and P100 fractions. Immunoblot analysis with antiserum to Aut1p indicated that Aut1p was localized to the supernatant fractions in both starvation conditions (Fig 5 C) and vegetative growth conditions (data not shown). We also attempted to follow localization of Aut1p by fusing it to GFP. However, neither NH2- nor COOH-terminal GFP fusions to Aut1p complemented the prAPI defect in the aut1 strain, and therefore were not further analyzed (data not shown). These findings demonstrate that Aut1p is predominantly a cytosolic autophagy component whose function is required for Aut7p membrane localization.
Aut1p, 2p, and 7p Are Involved in Autophagosome Vesicle Formation
Thus far, we have characterized Aut1p and Aut2p in terms of their distinct roles in Aut7p membrane association. We next examined the function of these components in the context of known steps in the transport of prAPI via the Cvt/autophagy pathways. Upon synthesis in the cytosol, prAPI oligomerizes (
To determine the step of the pathway in which Aut1p and Aut2p are required, we analyzed the state of prAPI in the null strains by protease sensitivity assays and membrane flotation gradients. The aut1 and aut2
strains were grown to midlog phase and converted into spheroplasts. After osmotic lysis of the spheroplasts, the lysates were separated into supernatant and pellet fractions by a 5,000 rpm centrifugation procedure. Under these conditions, prAPI appeared entirely in the pellet fraction (Fig 6). The prAPI-bound pellet fraction was subjected to exogenous protease treatment in the presence or absence of detergent. The protease sensitivity assays indicated that prAPI in the aut1
and aut2
strains was accessible to exogenous proteases even in the absence of detergent treatment (Fig 6), suggesting that prAPI in these strains was not protected by a completed vesicle.
In the membrane flotation gradient analysis, membrane-associated proteins migrate to the top of a step gradient and are collected as the "float" (F) fraction (see Materials and Methods). The prAPI-containing pellets in the aut1 and aut2
strains were subjected to flotation step gradients. The collected float fractions from both the aut1
and aut2
strains contained prAPI. However, treating the pellets with detergent prevented prAPI from floating through the gradient, suggesting that the buoyant population of prAPI was membrane associated (Fig 6). The aut7
and ypt7
strains were included in these analyses as controls. Ypt7p is a rab guanosine triphosphatase required for vesicle fusion with the vacuole and thus the ypt7
strain accumulates prAPI in a protease-protected, membrane-enclosed state (
strain is membrane associated (
The Apg Conjugation System Functions to Recruit Aut7p to the Membrane
In addition to the interactions between Aut7p and Aut2p ( control strain, a population of GFPAut7p was concentrated to punctate structures in the perivacuolar region (Fig 7 A), similar to the GFPAut7p pattern observed in the apg1
strain (Fig 3). However, in the apg5
, apg7
, and apg12
conjugation mutants, no punctate structures were detected, as GFPAut7p appeared diffusely localized in the cytosol (Fig 7 A).
Localization of Aut7p in the Apg conjugation strains by subcellular fractionations supports the confocal microscopy observations (Fig 7 B). The apg5, apg7
, and apg12
conjugation mutants and the apg9
control strain were grown to midlog phase and shifted to SD-N medium for 2 h before the subcellular fractionation procedure, which separated the lysates into S13, P13, S100, and P100 fractions. Immunoblot analysis of these samples revealed that Aut7p from the conjugation mutants appeared entirely in the supernatant fractions, along with the PGK cytosolic marker, while a significant population of Aut7p binds to the P13 fraction from the apg9
control strain (Fig 7 B). We also examined the Aut7p fractionation pattern in the apg10 and apg16
conjugation mutants. Apg10p acts at an intermediate step in the Apg12p-Apg5p reaction, while Apg16p mediates the dimerization of the final Apg12p-Apg5p conjugate (Fig 9 A;
strains appeared identical to that of the apg5
, apg7
, and apg12
strains (data not shown). These confocal microscopy and subcellular fractionation findings support the role of the Apg conjugation system in mediating Aut7p binding to a punctate, perivacuolar membrane compartment.
Aut7p Processing in the Conjugation Mutants and Apg Conjugation in the aut1 and aut2 Mutants
Because Aut1p and the Apg conjugation system are both required for Aut7p binding, we examined whether the defect in aut1 and the Apg conjugation mutants was at the step of Aut7p COOH-terminal proteolytic processing. While Aut7pGFP remained intact in the aut2
background, the fusion protein was cleaved in both the aut1
and Apg conjugation mutants (Fig 8 A). This finding indicates that although Aut7p membrane binding requires both Aut1p and the Apg conjugation system, the Aut7p COOH-terminal processing event occurs independently of their function. Conversely, Aut1p and Aut2p may be required for the Apg conjugation reaction, which involves the formation of a thioester bond between Apg12p and Apg7p before the transfer of Apg12p to Apg5p (
, aut2
, apg7
, and wild-type cells were transformed with a plasmid encoding an HA-tagged Apg5p fusion protein (
and aut2
cells, immunoblot analysis with antiserum to HA detected Apg5HAp in both the free form and as part of the Apg12pApg5p conjugate. This result indicates that Aut1p and Aut2p are not required for the Apg conjugation reactions (Fig 8 B). As expected, the Apg12pApg5p conjugate did not form in the apg7
strain.
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Discussion |
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A key early step in the membrane expansion and autophagic vesicle-formation processes may be defined by the recruitment of starvation-induced components to a membrane compartment involved in the formation of autophagic vesicles. The Aut7 protein is the first characterized transport vesicle component that is upregulated during starvation conditions ( strain defective for proteolytic processing (Fig 1 D). Similarly, a GFPAut7p fusion protein that functions as an in vivo marker for the autophagy pathway displays a diffuse cytosolic staining pattern in the aut2
mutant (Fig 3). In contrast, in most of the autophagy mutants that possess the capacity to process Aut7p (e.g., apg1
and apg9
; Fig 3 and Fig 7 A, respectively), the GFPAut7p fusion protein localizes to prominent, punctate perivacuolar structures in addition to its cytosolic distribution. These punctate structures may represent a previously described intermediate compartment that appears during autophagosome formation (
To further understand the role of Aut2p in the Aut7p cleavage reaction, we examined the Aut2 protein. Both subcellular fractionations (Fig 4 B) and confocal microscopy of the Aut2pGFP fusion protein (data not shown) indicate that Aut2p is a cytosolic, soluble protein. These findings indicate that the Aut2p-mediated cleavage of Aut7p most likely occurs in the cytosol. In addition, the observation that Aut2p expression does not increase under starvation conditions (data not shown) suggests that Aut2p is not a stoichiometric partner of Aut7p, but, rather, interacts with Aut7p in a catalytic manner to facilitate Aut7p proteolysis. Recently, background confirm these observations.
The recent two-hybrid analyses of the S. cerevisiae genome have presented an array of interactions between Apg components ( strain and in null mutants defective in the conjugation reaction. Both subcellular fractionations of the endogenous Aut7 protein and confocal microscopy analysis of the GFPAut7p fusion protein indicate that Aut1p and the Apg conjugation system are required for Aut7p membrane recruitment. Aut7p appears exclusively in the soluble, supernatant fractions in the aut1
and Apg conjugation mutants, while a significant amount of Aut7p binds to a low-speed pellet fraction in the other apg mutant strains (Fig 5 A and 7 B, respectively). In agreement with the fractionation results, the GFPAut7p fusion protein is diffusely distributed in the cytosol in the aut1
and in all of the Apg conjugation mutant strains. These findings establish a role for Aut1p and the Apg conjugation system in mediating Aut7p recruitment to an intermediate, punctate membrane compartment during autophagosome formation. Interestingly, the requirement of the conjugation component Apg16p, which facilitates the multimerization of the already-conjugated Apg12pApg5p species, suggests that it is not the conjugation reaction per se that plays a role in membrane binding of Aut7p, but rather some function of the multimeric Apg conjugation complex. Finally, as stated above, two-hybrid studies link Aut7p with Apg7p. It is interesting to note that the fluorescent staining pattern of GFPAut7p is similar to that of Apg7pGFP (
Although Aut1p interacts with Apg7p and Apg12p of the conjugation system, defects in Aut1p do not prevent the final conjugation of Apg12p to Apg5p, indicating that Aut1p is not directly required for the Apg conjugation reactions (Fig 8 B). Whether Aut1p and the conjugation system act in concert or independently through parallel pathways to recruit Aut7p to the membrane remains to be determined. However, neither Aut1p nor the Apg conjugation components are required for the Aut2p-mediated processing of Aut7p (Fig 8 A). Taken together, these findings suggest that Aut1p and the conjugation system act at a post-processing step in Aut7p membrane recruitment.
Other recent studies have offered two views of Aut7p function. An initial study by strain existed in a membrane-bound, protease-accessible state, suggesting that Aut7p was required at a step before vesicle completion. Our results indicate that prAPI in the aut1
and aut2
strains also exists in a membrane-associated but protease-accessible state, identical to the aut7
prAPI phenotype (Fig 6). Because Aut1p and Aut2p are required for Aut7p function, the common prAPI phenotype exhibited by the aut1, aut2, and aut7 null mutants supports the view that Aut1p, Aut2p, and Aut7p all act at the same step(s) of vesicle formation and/or completion.
The findings from this study can be incorporated into the current model of prAPI import by the Cvt and autophagy pathways (Fig 9 B). Immediately after synthesis, prAPI oligomerizes into a dodecamer, and then assembles into a higher-order Cvt complex. The Cvt complex then associates with a membrane of unknown origin. Interestingly, while deletions of AUT1, AUT2, and the Apg conjugation apparatus all prevent Aut7p membrane binding, prAPI in all cases retains its ability to bind to a pelletable membrane fraction (Fig 6). While these autophagy components are not required for prAPI membrane binding, additional factors may play a role in this process. The Aut2p-mediated cleavage of Aut7p generates a binding-competent form of this protein. However, both Aut1p and the Apg conjugation components are essential in facilitating Aut7p membrane binding. Our previous results indicated that the initial binding of Aut7p results in a protease-accessible form of the protein (
The Cvt and autophagy pathways share a common set of molecular components. However, the autophagosomes that are formed under starvation conditions are much larger than their Cvt vesicle counterparts formed under vegetative conditions. These observations suggest that formation of the autophagosome requires an expansion of the enwrapping membrane, as well as the increased expression of structural components. Aut7p may represent one member in a family of such structural components localized to these autophagic transport vesicles. Accordingly, the level of Aut7p expression in starvation versus vegetative conditions appears to reflect the 1020-fold increase in vesicle surface area between autophagosomes and Cvt vesicles (
In this study, we have characterized three events that are required for Aut7p recruitment to the membrane, a critical step in the formation of autophagic vesicles. In addition, these data define a function of the Apg conjugation system in recruiting structural components to the forming autophagosome. Further studies will be aimed at determining the specific modification effected by the conjugation machinery, the function of Aut1p in membrane binding of Aut7p, and the source of the membrane compartment to which Aut7p is recruited. These studies will begin to provide detailed mechanistic information about the formation of the sequestering vesicle used in the Cvt and autophagy pathways.
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Footnotes |
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1 Abbreviations used in this paper: Apg, autophagy; API, aminopeptidase I; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent protein; HA, hemagglutinin; ORF, open reading frame; PGK, phosphoglycerate kinase; SMD, synthetic minimal medium; TEA, tetraethylammonium.
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Acknowledgements |
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The authors thank Chao-Wen Wang for technical assistance, Dr. Neta Dean for her gift of the ME3 plasmid, and Drs. Hagai Abeliovich, Benjamin Lin, Sidney Scott, and Sarah Teter for helpful advice and critical reading of the manuscript.
This work was supported by Public Health Service grant GM53396 from the National Institutes of Health to D.J. Klionsky.
Submitted: 20 July 2000
Revised: 13 November 2000
Accepted: 17 November 2000
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References |
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