Yeast strains and growth media.
The yeast strains used in this study are listed in . BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 MET15 ura3Δ0) and the yeast deletion strain library were gifts from Greg Payne (University of California, Los Angeles [UCLA]). Proteasome-deficient (pre1-1 pre2-1) yeast strains were gifts from Dieter H. Wolf (University of Stuttgart, Stuttgart, Germany). The SEC7-6×dsRed (I33) TRP yeast strain was generously provided by Daniel Klionsky (University of Michigan). Cells were routinely grown in rich medium (yeast extract-peptone-dextrose [YPD]; Difco Chemicals, St. Louis, MO; 1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal dropout medium (SMD) (Difco Chemicals, St. Louis, MO; 0.67% yeast nitrogen base [YNB], 2% glucose, and specific amino acids) at 30°C unless otherwise stated. For galactose induction of green fluorescent protein (GFP) fusion reporters, cells were grown in synthetic minimal medium without uracil (SM-URA) and then shifted to SM-URA containing 0.2% galactose, 1% glycerol, and 1% ethanol. The ENV7-GFP strain was purchased from Invitrogen and confirmed by colony PCR.
All restriction enzymes and Taq DNA polymerase were purchased from New England BioLabs, Inc. (Beverly, MA). Phusion DNA polymerase was purchased from Invitrogen. Oligonucleotides were ordered from Operon (Alameda, CA). MG132 was supplied by EMD Chemicals. [γ-32P]ATP was purchased from MP Biomedicals (Solon, OH). Miniprep DNA kits, kits for gel extraction of DNA, and yeast transformation kits were supplied by Zymo Research (Irvine, CA). Antihemagglutinin (anti-HA), anti-hexokinase I, and anti-rabbit IgG–horseradish peroxidase (HRP)-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-alkaline phosphatase (anti-ALP) and anti-CPY antibodies were purchased from Mitosciences (Eugene, OR). HRP-conjugated anti-mouse goat monoclonal antibody was from Invitrogen. Anti-Vps41 antibody was a gift from Christian Ungermann (University of Osnabrück, Osnabrück, Germany). Chemiluminescence reagents were purchased from Pierce, and Film Biomax Light 13X18 PK50 was supplied by VWR. All other chemical reagents were from Sigma-Aldrich (St. Louis, MO).
Construction of plasmids and yeast transformation.
Yeast expression plasmids (listed in ) were constructed using established methods of PCR-directed homologous recombination (32
). Briefly, tagged versions of the ENV7
gene were PCR amplified from a genomic DNA using forward and reverse primers containing sequences (~45 bp) that are homologous to each end of the target vector to be cloned. The primers were ordered from Operon (Huntsville, AL) and are listed in . Cells were cotransformed with a linearized vector (containing a selectable marker) and a PCR-generated insert using a yeast transformation kit (Zymo Research, Irvine, CA), and the transformants were selected by growing on SM-URA agar plates. The constructed plasmids were isolated from yeast and transformed into Escherichia coli
. Plasmids with correct inserts were transformed into yeast with the desired background for functional analyses. HA- and GFP-tagged vectors were obtained from Walter Schmidt and used for construction of plasmids for expression of ENV7
-HA and GFP-tagged ENV7
Oligonucleotides used to construct plasmids
Construction, expression, and purification of His-tagged Env7 from yeast.
6×His-tagged Env7 was PCR generated using a pair of primers listed in , transformed into env7Δ yeast, and constitutively expressed under the phosphoglycerate kinase (PGK) promoter (PPGK), and the recombinant Env7-His was purified from BY4742 env7Δ according to the company protocol (Novagen). Briefly, yeast cells transformed with the 2μm plasmid (overexpressing C-terminally 6×His-tagged Env7) were grown to mid-log phase (optical density at 600 nm [OD600] = 1.0), harvested, and spheroplasted by Zymolyase treatment. The spheroplasts were broken open by bead beating with lysis buffer containing 1% Triton X-100 (TX-100) and subjected to high-speed centrifugation (28,000 × g for 1 h) using an Eppendorf centrifuge. The soluble fraction was resuspended with nitrilotriacetic acid (NTA)-Sepharose beads and eluted after several washes. The eluted protein was directly used for kinase assay.
Cloning, expression, and purification of His-tagged Env7 from E. coli.
A PCR product of C-terminally 6×His-tagged ENV7 was digested with NcoI and HindIII restriction enzymes and inserted into a pET24d(+) expression vector digested with the same enzymes, expressed in E. coli BL21(DE3)(pLysS) with 1 mM isopropyl-β-d-thiogalactoside (IPTG), and purified using Ni-Sepharose column chromatography according to the standard protocol as described by the company (Novagen).
PCR-based cloning was also carried out to insert DNA sequences of ENV7 into the 2μm overexpression vector pRS426. The ENV7 open reading frame (ORF) was amplified from BY4742 genomic DNA using custom-made forward and reverse primers (Operon, Huntsville, AL) that introduced restriction sites (underlined in ).
Single or multiple base substitutions in the target gene were introduced by using two PCR-based site-directed mutagenesis approaches as described previously (33
). All plasmid constructs and mutageneses were confirmed by DNA sequencing (Macrogen, South Korea).
For localization studies of endogenously expressed GFP-tagged Env7, yeast cells purchased from the Yeast GFP Clone Collection (Invitrogen) were grown in YPD to mid-log phase and analyzed by differential interference contrast (DIC) optics and confocal microscopy. An inducible GFP-Env7 or Env7-GFP reporter was constructed as described previously (34
). Mid-log-phase yeast cells containing the appropriate reporter were harvested, washed twice with sterile H2
O, and incubated in SM-Ura containing 0.2% galactose for approximately 5 h at 30°C to induce expression of the GFP reporters. The expression pattern of GFP reporters was visualized using a confocal microscope as described previously (24
). The galactose-induced cells were viewed with an Olympus Fluoview 1000 confocal laser scanning system mounted on an inverted microscope (Olympus IX-81) and a 100× oil immersion UPLSAPO objective (numeric aperture [NA], 1.4; working distance [WD], 0.12 mm). The Argon ion (488-nm) and blue/red diode (405-nm/635-nm) lasers were used for image capturing. Images were equally enlarged to ×4,000 and were analyzed and processed with Photoshop CS5. For each experiment, at least 150 to 200 cells from five visual fields were scored, from which representative images were selected. Images for colocalization studies were created by overlapping the DIC and fluorescence images.
For vacuole morphology studies, log-phase cells were stained with the vital dye FM4-64 [N
-diethylaminophenylhexatrienyl)pyridiumdibromide] (Invitrogen) as described previously (24
). For hyperosmolarity studies, stained cells were subjected to hyperosmotic shock (0.4 M NaCl) for 15 and 60 min and analyzed microscopically as described above. One hundred to 150 cells were blind scored from representative microscopic fields for statistical analysis of vacuolar morphology (prominent versus multilobed vacuoles). Similarly, stained cells were also scored for bud vacuolar morphology (prominent versus multilobed vacuoles) under normal growth conditions. For vacuole acidification studies, log-phase cells were stained with Quinacrine (Invitrogen) for 5 min and analyzed microscopically as described previously (35
Subcellular fractionation of yeast cells was performed as described previously (36
) with the following modifications. The homogenate (H) was centrifuged twice at 750 × g
for 10 min to remove unbroken cells, cell debris, or nuclei. The clarified supernatant (termed the postnuclear fraction [PNF]), was further centrifuged at 18,000 × g
for 50 min to obtain a membrane-rich pellet (P18) and clear cytosol (S18). The membranous fraction was then resuspended in 0.1 M sodium carbonate (pH 11.5), incubated on ice for 30 min, and centrifuged at 18,000 × g
for 30 min. The protein concentration was determined by the Bradford method as described below and adjusted to 0.8 mg/ml. For subcellular localization studies of the triple-cysteine (C13-15S) mutant Env7-HA, yeast cells were lysed and subjected to differential centrifugation to yield S0.4, P13, P100, and S100 fractions as described previously (20
Ficoll gradient isolation of vacuoles.
Yeast vacuoles were isolated as described by Haas and Wickner (37
). Interface fractions were trichloroacetic acid (TCA) precipitated, and the pellets were washed 3 times with cold acetone and resuspended in 6× SDS-PAGE sample buffer. Solubilized fractions were separated on 12% SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by Western blotting.
Functional complementation assay.
To confirm in vivo
functionality of N- and C-terminally GFP-tagged, HA-tagged, and overexpressed untagged Env7, the constructed plasmids were introduced into wild-type and env7
Δ yeast strains. Transformed cells were selected on SM-URA and were subjected to patch immunodetection as previously described (24
Bioinformatic and phylogenetic analyses.
For hydrophobicity/hydrophilicity analysis, the Env7 sequence was copied from the Saccharomyces
Genome Database (SGD) and pasted into the analysis window hosted by the Genome Consortium for Active Teaching (GCAT) at Davidson College (http://gcat.davidson.edu/rakarnik/kyte-doolittle.htm
), and a Kyte-Doolittle analysis (38
) was performed on the entire sequence using an amino acid window size of 19.
For multiple-sequence alignment (MSA), the ENV7
nucleotide sequence query was used in a tBLASTx search to determine putative homologs (39
). Default search settings were utilized, and entire sequences with the lowest E value (an upper limit of 1e−10
) from each species were chosen for further analysis. Thirty-one nucleotide sequences (including ENV7
) were aligned using the MAFFT multiple-sequence translation alignment program (40
). The E-INS-I alignment algorithm and a BLOSUM80 scoring matrix were employed, and a gap open penalty of 2 was used with no offset value. The program output alignment was screened and edited manually.
For phylogenetic analyses, the alignment was submitted to MrBayes, with the Zea mays
sequence as the outgroup (41
). The substitution model utilized was general time reversible (GTR) with a gamma rate variation and four gamma categories. One million iterations were generated with a burn-in length of 250,000. Branch lengths were unconstrained. The results were visualized with Geneious and Dendroscope (42
). The tree topology was assessed for congruence with a maximum-likelihood tree constructed from ATP synthase homologs using the online program Icong
The palmitoylation assay was carried out according to a biotinylation assay method, as described previously (46
) with modifications. Briefly, P18 membranes were resuspended in ABE buffer (50 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 1% Triton X-100, and protease inhibitors) and immunoprecipitated overnight using 10 μl anti-HA rabbit monoclonal antibodies conjugated to Sepharose beads (Cell Signaling Technology, Danvers, MA). The beads were washed with ABE buffer containing 0.1% Triton X-100 and incubated with 50 mM N
-ethylmaleimide (NEM) in ABE buffer for 1.5 h. They were washed and incubated with either 1 M Tris, pH 7.5 (control), or 1 M hydroxylamine, pH 7.5, for 2.5 h at room temperature. The beads were then incubated overnight with 300 μM biotin and resuspended in sample buffer. An aliquot was separated by SDS-PAGE and analyzed by Western blotting using streptavidin HRP (for biotinylation) and anti-HA antibody (for protein recovery).
For kinase assays of Env7-HA, the whole membranous fractions (P18) were used. The membranes were solubilized by resuspending them in lysis buffer containing 1% Triton X-100 and 500 mM NaCl, incubated on ice for 30 min, and centrifuged at 18,000 × g for 30 min to obtain solubilized Env7-HA, which was then immunoprecipitated overnight at 4°C as described above. The Sepharose beads were washed 3 times in lysis buffer (containing 1% Triton X-100, 500 mM NaCl), once in 1 M NaCl, and 3 times in kinase buffer (0.05 M Tris-HCl, pH 7.4, containing 10 mM MgCl2 and 2 mM dithiothreitol [DTT]) and used in kinase assays. Kinase activity was assayed in a total reaction volume of 20 μl that contained 10 μl of immunoprecipitated beads (Env7-HA), 200 μM unlabeled ATP, 1 mg/ml bovine serum albumin (BSA), and 10 μCi [γ-32P]ATP with or without the addition of 2.5 μg exogenous substrate. For some experiments, the detergent-solubilized residual membranes were also used for kinase assay. The kinase reaction was carried out by incubating the reaction mixture at 30°C for 30 min and stopped by the addition of 5 μl of 6× SDS-PAGE sample buffer. The solubilized proteins were heated at 100°C for 10 min and resolved by 12% SDS-PAGE. The gels were fixed (10% acetic acid, 40% methanol) and stained with Coomassie brilliant blue R250 (CBB), and the dried gels were subjected to autoradiography BioMax film with maximum resolution (Kodak, Rochester, NY).
Vps41 phosphorylation assay.
The Vps41 phosphorylation assay was based on upshift of phosphorylated Vps41 migration in SDS-PAGE and was performed using the P13 membrane fraction and anti-vps41 antibody as described previously (20
Western blot analysis.
Western blots were carried out using a semidry method with appropriate primary and secondary antibodies, as previously described (24
Protein concentrations were determined by the Bradford method (48
) using the Quick Start Bradford protein assay kit (Bio-Rad, Hercules, CA). Protein concentrations were calculated from a linear standard curve of BSA (plotted from a graph of absorbance versus protein concentrations ranging between 0 and 1 mg/ml) using Microsoft Excel.
For vacuolar morphology studies, 150 to 200 cells were blind scored from random fields in at least two separate experiments, and their mean values and standard deviations were calculated using standard statistical tools (Excel). P values were calculated using a t test. P values of <0.05 were considered statistically significant.