The yeast strains used in this study are listed in .
Yeast Strains Used in this Study
To prepare antiserum to Apg9p/Cvt7p, synthetic peptides corresponding to amino acids 1–15 (National Institute for Basic Biology, Center for Analytical Instruments) or 74–89 and 138–156 (Multiple Peptide Systems) were synthesized and conjugated individually to BSA or keyhole limpet hemocyanin, respectively. Standard procedures were used to generate antiserum in Japanese White or New Zealand White rabbits, respectively. Antisera against API and carboxypeptidase Y (CPY) were prepared as described previously (Klionsky et al. 1988
, Klionsky et al. 1992
). Antiserum against phosphoglycerate kinase (PGK) was generously provided by Dr. Jeremy Thorner (University of California, Berkeley, CA; Baum et al. 1978
). Anti-HA mAb 16B12 was from Berkeley Antibody Co., Inc. FITC-conjugated secondary anti-mouse antibody was from Roche Molecular Biochemicals.
All restriction enzymes and VentR DNA polymerase were from New England Biolabs. AmpliTaq DNA polymerase was from Perkin Elmer Biosystems. Expre35S35S Protein Labeling Mix was from Dupont-New England Nuclear Research Products. Immobilon-P (polyvinylidene fluoride, PVDF) was from Millipore. ConA Sepharose and Ficoll-400 were from Pharmacia Biotech, Inc. Complete™ EDTA-free protease inhibitor cocktail and endoglycosidase H (endo H) were from Roche Molecular Biochemicals. FM 4-64 dye was from Molecular Probes. Oxalyticase was from Enzogenetics. YNB was from DifCo Laboratories, Inc. Zymolyase 100T was from Seikagakukogyo. Oligonucleotides to pBR322 were from Promega. All other oligonucleotides were synthesized by Operon Technologies, Inc. All other reagents were from Sigma Chemical Co. or Wako Chemicals.
Yeast cells were grown in rich medium (YPD; 1% yeast extract, 2% peptone, and 2% glucose) or various synthetic minimal media. Synthetic minimal media with carbon and nitrogen sources were: SMD, 0.67% yeast nitrogen base, 2% glucose, and auxotrophic amino acids and vitamins as needed; SD, 0.17% yeast nitrogen base without ammonium sulfate and amino acids, 0.5% ammonium sulfate, and 2% glucose; or SCD, 0.17% yeast nitrogen base without ammonium sulfate and amino acids, 0.5% ammonium sulfate, 0.5% casamino acids and 2% glucose. Synthetic minimal media lacking nitrogen and/or carbon sources were: SD(−N), 0.17% yeast nitrogen base without ammonium sulfate and amino acids and 2% glucose; and S(−N, −C), 0.17% yeast nitrogen base without ammonium sulfate and amino acids.
Isolation of Mutants
mutants were isolated on the basis of prAPI accumulation (Harding et al. 1995
, Harding et al. 1996
belongs in the group of mutants that share a genetic overlap with mutants defective in autophagy, and is in the same complementation group as aut9
(Harding et al. 1996
; Scott et al. 1996
). The cvt7-1
mutant was used for subsequent analyses and cloning of the CVT7
gene as described below.
The Pho8Δ60p construct (Noda et al. 1995
) was used to screen for additional autophagy-defective mutants. Pho8Δ60p is an altered form of the vacuolar hydrolase alkaline phosphatase lacking the NH2
-terminal transmembrane domain. This modified protein is only delivered to the vacuole through autophagy (). Proteolytic cleavage of the Pho8Δ60p propeptide in the vacuole lumen generates the active form of the enzyme. Yeast strain TN124 expressing Pho8Δ60p was mutagenized with EMS and spread onto YPD plates. Approximately 36,000 colonies were replica-plated onto SD(−N) and incubated overnight at 30°C. 1% agar containing 0.1 mM α-naphthyl phosphate, 1 mg/ml Fast Red TR salt, 5 mM MgCl2
, 0.1 M Tris-HCl, pH 9.0, and 1 mM PMSF was melted and kept at 60°C. Fast Red TR salt and PMSF were added to the melted agar, and 5 ml of this mixture was poured onto the plate. Colonies that were able to carry out autophagy and exhibited alkaline phosphatase activity turned brown. The colonies that did not turn brown were picked from the master plate and were subjected to a morphological observation of autophagy. Mutants that did not accumulate intravacuolar vesicles upon starvation in the presence of PMSF (Tsukada and Ohsumi 1993
) were placed into complementation groups. The most abundant group was found to fit into the complementation group that was previously identified as apg9
(Tsukada and Ohsumi 1993
). The apg9-1
mutant was chosen for further analysis.
Cloning of CVT7/APG9
gene was independently cloned by our two laboratories. Because >99% of apg9/cvt7
cells die after 6 d of nitrogen starvation, the APG9/CVT7
gene product is essential for cell viability under autophagy-inducing conditions of nitrogen deprivation. Therefore, survival in nitrogen-poor medium offered a convenient selection strategy to clone APG9/CVT7
. Specifically, strain THY154 (cvt7-1
) and CT83-1B (apg9-1
) cells were transformed with a YCp50-based yeast genomic library (Rose et al. 1987
). Approximately 60,000 transformed colonies were then subjected to nitrogen starvation for 6 d. Transformants that survived this regimen were subjected to Western blot analysis. Transformants were isolated containing genomic plasmids that complemented the prAPI processing defect of cvt7-1
or the ability to accumulate autophagic bodies in apg9-1
. Partial sequences from these genomic plasmids were entered into the Saccharomyces
Genome Database (SGD; http://genome-www.stanford.edu/Saccharomyces/). The full-length sequences of these plasmids contained overlapping fragments of 7–8 kb. After further manipulations, the open reading frame (ORF) of APG9/CVT7
was identified as YDL149w. For clarity, we will refer to the APG9/CVT7
gene and the gene product as APG9
and Apg9p, respectively, henceforth in this section.
Using a NotI/BamHI digestion, an APG9 cassette with 757 bp of upstream and 485-bp downstream sequences was subcloned into pRS414 and pRS424, resulting in plasmids pAPG9(414) and pAPG9(424), respectively. Both pAPG9(414) and pAPG9(424) complemented the prAPI processing defect when transformed into the cvt7 and apg9 mutants. Similarly, a 4.8-kb fragment containing a part of YCp50 cut with XbaI from plasmid p8001 encoding APG9 was subcloned into pRS316 to generate plasmid p8007. This plasmid was sufficient to suppress the autophagy defect of apg9.
Disruption of the APG9 Chromosomal Locus
The chromosomal APG9 locus was disrupted through two independent approaches. First, a PCR-based strategy was employed to disrupt the chromosomal copy of APG9. The HIS3 gene was PCR-amplified from the pRS413 vector using oligonucleotides containing vector sequences outside of the HIS3 auxotrophic marker flanked by APG9 sequences encoding regions at the beginning and end of the APG9 ORF: 5′-GAGAGAGATGAATACCAGTTACCCAACTCTCATGGGAAGTTGTAC-TGAGAGTGCACCAT-3′ and 5′-GGATGATGTACACGACACAGTCTGCCTTATCTTCCGACGTCGGTATTTCACACCGCATA-3′. The PCR product was transformed into strain SEY6210 wild-type cells and grown on minimal plates lacking histidine. Transformants were then assayed for API precursor accumulation. In addition, the apg9Δ strain was crossed with the cvt7-1 mutant, followed by tetrad analysis and Western blotting with API antiserum. All of the germinants from 12 tetrads exhibited a prAPI phenotype indicating that the APG9 gene maps to the correct locus. The apg9Δ strain was named JKY007.
In the second approach, plasmid p8007 was digested with EcoRI to remove a 700-bp fragment within the APG9 ORF. An EcoRI fragment containing the TRP1 gene was ligated into the EcoRI sites of p8007 to generate p8009. Plasmid p8009 was digested with EcoT14I/SphI and a fragment containing the TRP1 marker and flanking APG9 sequences was transformed into YW5-1B wild-type cells. Disruption at the APG9 locus was confirmed by Southern blot. The apg9Δ strain was crossed with the apg9-1 mutant. The resulting diploid retained an autophagy defect suggesting that APG9 is YDL149w.
Cell Viability Under Nitrogen Starvation Conditions
To examine the survival of various yeast strains under nitrogen starvation conditions, cells were grown to an OD600 = 1 in SMD, washed in SD(−N), and then resuspended in SD(−N) to an OD600 = 1. At the indicated times, aliquots were removed and plated onto YPD plates in triplicate. Colonies that survived the nitrogen starvation regimen were counted after 2–3 d.
Construction of Plasmids
For epitope-tagging Apg9p, a BamHI site was generated next to the initiation methionine of APG9 in plasmid pRS424 or pRS426. A DNA fragment encoding a 3×HA epitope with BamHI sites on both sides was then ligated into the BamHI site.
To construct the APG9GFP
centromeric and multicopy plasmids, the APG9
gene, including 509 bp of upstream sequence, was PCR-amplified from the pAPG9(414) template using oligonucleotides that incorporated a NotI site in the 5′ primer (5′-GCAAGGTTGGGTCTATGCGGCCGCACAAATCTGG-3′) and an in-frame BamHI site immediately preceding the APG9
stop codon in the 3′ primer (5′-GACACAGTCTGCCTTAGGATCCGAC-3′). The 3.5-kb PCR product was subcloned into pCGFP(416) and pCGFP(426), which contained the green fluorescent protein (GFP) ORF, followed by actin termination sequences (Kim et al. 1999
). The resulting COOH-terminal GFP fusion constructs, pAPG9GFP(416) and pAPG9GFP(426), were tested for their ability to rescue the prAPI import defect in the apg9
For cell labeling experiments, cells were grown to OD600
= 1 and resuspended in SMD at 20–30 OD600
/ml. The resuspended cells were labeled with 10–20 μCi of 35
S Express label/OD600
for the indicated times, followed by a chase reaction in SMD supplemented with 0.2% yeast extract, 4 mM methionine, and 2 mM cysteine at a final cell density of 1 OD600
/ml. The labeled cells were precipitated with 10% TCA on ice, followed by two acetone washes. Crude extracts were prepared by glass bead lysis, as described previously (Harding et al. 1995
Wild-type cells were grown to midlog and incubated in 0.1 M Tris-HCl, pH 7.5, containing 40 mM 2-mercaptoethanol for 10 min at room temperature. Following a centrifugation step, the cells were spheroplasted in 1.2 M sorbitol, 50 mM Tris-HCl, pH 7.5, with 5 U/ml Zymolyase 100T at 30°C for 30–50 min. The cells were loaded on top of an equal volume of 1.8 M sorbitol and centrifuged at 2,000 rpm for 10 min. The spheroplasts were collected from the bottom of the tube. For sucrose density gradient fractionation, YW5-1B cells were grown to midlog in YPD and spheroplasted as described above. The spheroplasts were lysed in lysis buffer B (20 mM triethanolamine, pH 7.2, 12.5% sucrose [wt/vol], 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail) at a density of 60 OD600/ml. After removal of cell debris, the supernatant fraction (1 ml) was layered on top of a sucrose density gradient consisting of 1-ml steps of 54, 50, 46, 42, 38, 34, 30, 26, 22, and 18% sucrose (wt/wt) in 10 mM Hepes-NaOH, pH 7.5, 1 mM MgCl2, and 1 mM PMSF. The gradients were subjected to centrifugation in a Hitachi RPS40T rotor at 174,000 g for 2.5 h. Samples were collected into 16 fractions from the top of the gradients. To examine Apg9p in the ypt7Δ strain, the precleared lysate was subjected to centrifugation at 100,000 g for 1 h. The pellet was resuspended in lysis buffer B and centrifuged a second time at 100,000 g for 1 h. The pellet fraction after the second centrifugation step was resuspended in 1 ml of lysis buffer B and subjected to the same density gradient centrifugation as above for 16 h.
Endoglycosidase H Treatment
Strain CTD1 cells expressing 3×HA Apg9p were grown to midlog phase and converted to spheroplasts. The spheroplasts were lysed in 0.1% SDS, 50 mM sodium citrate, pH 5.5, 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail, and boiled for 5 min. After centrifugation at 14,000 rpm for 1 min, the protein concentration of the supernatant fraction was determined. Protein (60 mg) was diluted to 25 μl, and 25 μl 0.1% SDS, 0.1 M 2-mercaptoethanol were added, and the solution was boiled for 3 min. 15 μl of 0.5 M sodium citrate, pH 5.5, 75 μl H2O, 5 μl 100 mM PMSF, 2.5 μl endo H (1 U/ml) were then added and the mixtures were incubated overnight at 37°C, boiled for 3 min, and subjected to immunoblotting.
Generation of the apg9ts Mutant
A strategy of gap-repair PCR mutagenesis (Muhlrad et al. 1992
) was employed to generate a temperature conditional mutant of Apg9p. In brief, a “gapped” APG9
plasmid was created by digestion with SpeI/BglII, which removed a 1.74-kb piece of the APG9
ORF starting 228 bp downstream of the ATG start site. A 2.23-kb PCR product of the APG9
ORF was generated by using the primers 5′-CACGGAATTATTAGGTTATGGAGAGAGATG-3′ and 5′-GTGTTATCATCAGGCTAGTGAGTTCCC-3′ and pAPG9(414) as the template. Errors were introduced in the polymerization reactions by the addition of 0.1 mM MnCl2
. The resulting mutagenized PCR product shares overlapping sequences of 245 bp at the 5′ side and 240 bp at the 3′ side with the gapped APG9
plasmid. The PCR product and the gapped plasmid were cotransformed into the apg9
Δ strain and grown on −Trp plates. The transformants were subjected to Western blotting analysis with anti-API antiserum after growth on −Trp plates overnight at 25 or 37°C. Out of the 400 colonies examined, four colonies exhibited a mature API phenotype at 25°C, indicative of functional Apg9p, and a prAPI phenotype at 37°C, the defective Apg9p phenotype. The four potential apg9ts
colonies were analyzed by pulse/chase labeling to assess the kinetics of prAPI delivery to the vacuole at permissive and nonpermissive temperatures.
Membrane Flotation Analysis
The procedure for the membrane flotation experiments is a modification of a previously described protocol (Scott and Klionsky 1995
; Babst et al. 1997
; Kim et al. 1999
). Spheroplasts from the apg9ts
and the ypt7
Δ strains were pulse-labeled with Expre35
S label for 10 min, followed by a 30-min chase at 38°C. After centrifugation at 1500 g
for 5 min, the spheroplasts were lysed in osmotic lysis buffer C (20 mM Pipes, pH 6.8, 200 mM sorbitol, 5 mM MgCl2
, Complete EDTA-free protease inhibitor cocktail) at a spheroplast density of 20 OD600
/ml. The lysate from 16 OD600
cell equivalents was subjected to a 5,000 g
centrifugation for 5 min, resulting in low-speed supernatant (S5) and pellet (P5) fractions. The P5 fractions, which contained all of the prAPI, were resuspended in 100 μl of 15% Ficoll-400 (wt/vol) in gradient buffer (GB; 20 mM Pipes, 5 mM MgCl2
, Complete EDTA-free protease inhibitor cocktail) with or without the addition of 0.2% Triton X-100. The resuspended P5 fractions were overlaid with 1 ml of 13% Ficoll-400 in GB and then 300 μl of 2% Ficoll-400 in GB. The resulting step gradients were subjected to centrifugation at 15,000 g
for 10 min at 25°C in a microcentrifuge. Fractions were collected from the top. The first 500 μl was the float fraction, the remaining 900 μl was the nonfloat fraction, and the gradient pellet was considered the pellet fraction. The resulting fractions were TCA precipitated and washed twice with acetone before being immunoprecipitated with anti-API antiserum.
Protease Sensitivity Analysis
To examine the protease sensitivity of prAPI in the apg9ts and ypt7Δ strains, spheroplasts were labeled for 10 min at 38°C followed by a 30-min chase reaction at 38°C. Spheroplasts were lysed in osmotic lysis buffer C at a spheroplast density of 20 OD600/ml. The resulting lysate (20 OD600 cell equivalents per incubation condition) was separated into S5 supernatant and P5 pellet fractions by centrifugation at 5,000 g for 5 min at 4°C. The P5 pellet was resuspended in osmotic lysis buffer C in the presence or absence of proteinase K (50 μg/ml) and 0.2% Triton X-100. The resuspended P5 pellets were incubated on ice for 20 min, followed by TCA precipitation, acetone wash, and immunoprecipitation with anti-API and anti-PGK antisera.
Immunofluorescent Labeling, Immunoelectron Microscopy, and Confocal Microscopy
For immunofluorescent labeling of vegetatively growing cells, cultures were grown to midlog in YPD. To examine cells under starvation conditions, cells were grown in YPD and subsequently incubated in SD(−N) with 1 mM PMSF for 3 h. The cells were fixed in 100 mM potassium phosphate, pH 6.5, containing 5% formaldehyde for 2 h at 23°C. Following a washing step in SHA buffer (1 M sorbitol, 0.1 M Hepes-NaOH, pH 7.5, 5 mM NaN3), the fixed cells were converted to spheroplasts by incubation with 0.5 U/ml Zymolyase 100T, 24 mM 2-mercaptoethanol for 15 min at 30°C. The spheroplasts were washed in SHA buffer and mounted on polylysine-coated multiwell glass slides. The cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 min. Permeabilized cells were incubated with anti-HA mAb at a 1:5,000 dilution, and the FITC-conjugated secondary anti-mouse antibody was used at a 1:50 dilution. Photographs were taken using the confocal microscope Zeiss LSM510.
For EM, cells were subjected to rapid freezing and freeze-substitution fixation, and observed as described previously (Baba et al. 1997
). For immunoelectron microscopy, ultrathin sections were collected onto formvar-coated nickel grids and blocked in PBS containing 2% BSA at room temperature for 15 min. Incubations were carried out by floating grids on a 20 μl drop of a 1:1,500 dilution of anti-HA mAb, 16B12, at room temperature for 1.5 h. After washing, the grids were incubated for 1 h with 5-nm gold-conjugated goat anti–mouse IgG. The grids were washed several times in PBS followed by several drops of distilled water and fixed with 1% glutaraldehyde for 3 min. The sections were stained with 4% uranium acetate for 7 min and examined.
Strains expressing the Apg9pGFP fusion protein were grown to midlog and the vacuoles were labeled with FM 4-64 dye to a final concentration of 4–8 μM and incubated at 30°C for 20 min to allow cells to take up the dye. After a wash step, the cultures were resuspended in SMD for 2 h to allow the dye to label the vacuole via endocytosis. Cells were examined on a Leica DM IRB confocal microscope. The images captured were the result of an average of eight scans of a single focal plane.
Hydrophobicity analysis was performed using the program TopPred II developed by M.-G. Claros (Laboratoire de Génétique Moléculaire, Paris, France).
Immunoblots were scanned using a Power Macintosh G3 and GT-9000 scanner (EPSON), and band intensities were determined using the program NIH Image 1.61.1. Alternatively, autoradiograms were quantified using the Molecular Dynamics Storm PhosphorImager and ImageQuant 5.0.