Yeast strains used in this study are summarized in Table S1
. To obtain the GFP-tagged Grh1 in various deletions strains, the tagging was either induced by homologous recombination (Janke et al., 2004
) or the deletion strains (BY4741 background) were mated with the Grh1-GFP strain (Matα) and sporulated in 2% potassium acetate (supplemented with amino acids as needed) at 30°C. Spore preparation was performed as described previously (Herman and Rine, 1997
). Haploidity was confirmed using the halo mating type assay (Sprague, 1991
To generate the Grh1-GFP fusion protein under the control of its endogenous promoter, the coding sequence of Grh1, including 1 kb upstream of the start codon without the stop codon, was amplified by PCR and cloned as a ClaI–BamHI fragment into the pRS416 plasmid. Yeast EGFP was amplified by PCR from pyM26 and cloned as a BamHI–SacI fragment into the Grh1 containing pRS416 to generate the pRS416 Grh1-GFP plasmid. The coding sequence of Ape1 including its own promoter was amplified by PCR and cloned as a Sac1–Spe1 fragment into the pRS416 plasmid containing the mCherry coding sequence to generate the pRS416 Ape1-mCherry plasmid.
mCherry-Atg8 was cloned into pRS316 plasmid under the control of its endogenous promoter and was provided by Y. Ohsumi (National Institute for Basic Biology, Okazaki, Japan). RFP-Atg9 was cloned into the pRS416 plasmid under the control of the CupI promoter (Chang and Huang, 2007
) and provided by W.P. Huang (National Taiwan University, Taipei, Taiwan). Vps23-mCherry was cloned into pRS416 under the control of its endogenous promoter (Curtiss et al., 2007
) and provided by M. Babst (University of Utah, Salt Lake City, UT). mCherry-Tlg1 and mCherry-Pep12 were cloned into pRS416 under the control of the Vps21 promoter and provided by D. Katzmann (Mayo Clinic, Rochester, NY). Plasmids for the expression of Sec7-DsRed (pRS316 Sec7-DsRed; Calero et al., 2003
) and DsRed-FYVE (pRS425MET3 DsRed-FYVE; Katzmann et al., 2003
) were provided by S. Emr (Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY).
Yeast cells were grown in rich YPD (1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal media (synthetic complete glucose; 0.67% yeast nitrogen base, 2% glucose, amino acids, and vitamins as needed). Starvation conditions were induced by culturing yeast cells in 2% potassium acetate at 1 OD600nm
. Cycloheximide treatment was performed by adding 250 µg/ml cycloheximide to growth or starvation medium, and rapamycin treatment was performed by adding 0.4 µg/ml to the nutrient-rich medium for 3 h (Suzuki et al., 2002
). Unless otherwise indicated, cells were grown at 25°C.
Yeast cells were grown in the appropriate medium before imaging. Cells were harvested at 500 g for 3 min in a table top centrifuge (Multifuge 3 L-R; Heraeus), spotted on a microscopy slide, and immediately live imaged with a microscope (DMI6000 B; Leica) equipped with a camera (DFC 360FX; Leica) using an HCX Plan Apochromat 100× 1.4 NA objective. Images were taken using LAS AF software (Leica) with the same exposure times for Grh1-GFP; colocalization analysis with different marker proteins was performed with lower exposure times. Figures were assembled in Photoshop (Adobe) with only linear adjustments. Statistical analysis of the localization was performed by counting ≥60 cells from three independent experiments, and the statistical significance was tested in an unpaired Student’s t test using Prism (GraphPad Software). Compared data sets were termed as statistically significant when P < 0.05.
Staining of endocytic intermediates from endosomal to vacuolar membranes with the lipophilic dye FM 4-64 was performed as described previously (Vida and Emr, 1995
) with slight modifications. Cells were grown at 25°C to an OD600nm
of 0.8. A 10-ml culture was centrifuged and resuspended at 30 OD600nm
/ml. FM 4-64 (Invitrogen) was added at a final concentration of 30 µM, and cells were incubated for 15 min at 25°C. Cells were harvested by centrifugation, resuspended at 1 OD600nm
in starvation medium, and cultured at 25°C. At different time points, cells were collected by centrifugation, spotted on a slide, and live imaged.
Conventional and immunoelectron microscopy
Yeast cells expressing Grh1-GFP or Grh1-GFP and Sec13-RFP were grown in normal growth medium or starved for 4 h at 30°C. Preparation of samples for morphology and ultrathin cryosections as well as the immunoelectron microscopy was performed as described previously (Rieder et al., 1996
). In brief, for conventional electron microscopy, cells were pelleted and fixed in 3% glutaraldehyde contained in 0.1 M sodium cacodylate, pH 7.4, 5 mM CaCl2
, 5 mM MgCl2
, and 2.5% sucrose for 1 h at 25°C with gentle agitation, spheroplasted, embedded in 2% ultra low temperature agarose (prepared in water), cooled, and subsequently cut into small pieces (~1 mm3
). The cells were then postfixed in 1% OsO4
/1% potassium ferrocyanide contained in 0.1 M cacodylate/5 mM CaCl2
, pH 7.4, for 30 min at 25°C. The blocks were washed thoroughly four times with double-distilled H2
O; 10 min in total), transferred to 1% thiocarbohydrazide at 25°C for 3 min, washed in ddH2
O (four times for 1 min each), and transferred to 1% OsO4
/1% potassium ferrocyanide in cacodylate buffer, pH 7.4, for an additional 3 min at 25°C. The cells were washed four times with ddH2
O (15 min in total), en bloc stained in Kellenberger’s uranyl acetate for 2 h to overnight, dehydrated through a graded series of ethanol, and subsequently embedded in Spurr resin. Sections were cut on an ultramicrotome (Ultracut T; Reichert), poststained with uranyl acetate and lead citrate, and observed on a transmission electron microscope (Tecnai 12; FEI) at 100 kV. Images were recorded with a digital camera (Soft Imaging System MegaView III; Olympus), and figures were assembled in Photoshop with only linear adjustments in contrast and brightness.
For immunoelectron microscopy, cells were fixed in suspension for 15 min by adding an equal volume of freshly prepared 8% formaldehyde contained in 100 mM potassium phosphate buffer, pH 7.4. The cells were pelleted, resuspended in fresh fixative (8% formaldehyde and 100 mM PO4, pH 7.4), and incubated for an additional 18–24 h at 4°C. The cells were washed briefly in PBS and resuspended in 1% low gelling temperature agarose. The agarose blocks were trimmed into pieces of 1 mm3, cryoprotected by infiltration with 2.3 M sucrose/30% polyvinyl pyrrolidone (10,000 molecular weight)/PBS, pH 7.4, for 2 h, mounted on cryopins, and rapidly frozen in liquid nitrogen. Ultrathin cryosections were cut on an ultramicrotome (UCT; Leica) equipped with an FC-S cryoattachment and collected onto Formvar/carbon-coated nickel grids. The grids were washed through several drops of PBS containing 2.5% FCS and 10 mM glycine, pH 7.4, and then blocked in 10% FCS for 30 min and incubated overnight in chicken anti-GFP antibody (Abcam) or chicken anti-GFP plus rabbit anti-RFP (Abcam). After washing, the grids were incubated for 2 h in 12-nm gold donkey anti–chicken conjugate or a mixture of 12-nm donkey anti–chicken plus 6-nm donkey anti–rabbit (Jackson ImmunoResearch Laboratories, Inc.). The grids were washed through several drops of PBS followed by several drops of ddH2O. Grids were then embedded in an aqueous solution containing 3.2% polyvinyl alcohol (10,000 molecular weight)/0.2% methyl cellulose (400 centipoises)/0.1% uranyl acetate. The sections were examined and photographed on a transmission electron microscope (Tecnai 12) at 100 kV, and images were collected with a digital camera (Soft Imaging System MegaView III). Figures were assembled in Photoshop with only linear adjustment of contrast and brightness.
250 OD600nm of yeast expressing Grh1-GFP were grown either in normal growth medium or starved for 3 h. Cells were collected by a 5-min spin at 3,000 g and washed once with ice-cold 10 mM NaN3/NaF. Cells were resuspended at 20 OD/ml in prespheroplasting buffer (10 mM NaN3, 10 mM NaF, 100 mM Tris-H2SO4, pH 9.4, and 0.36 µl/ml β-mercaptoethanol) and incubated for 20 min at 25°C. Cells were collected and spheroplasted at 50 OD600nm/ml in spheroplasting buffer (40 mM Hepes-NaOH, pH 7.5, 1.4 M sorbitol, and 1 µl/ml β-mercaptoethanol) by treatment with 50 U/OD600nm Zymolyase for 45 min at 35°C. Spheroplasts were harvested, resuspended in 1.5 ml lysis buffer (10 mM Hepes-NaOH, 1 mM MgCl2, 0.3 M sorbitol, and protease inhibitors), and lysed by using a Dounce homogenizer (Afora). Lysates were cleared twice by centrifugation (600 g for 3 min). Equal protein concentrations were loaded on top of a continuous sucrose gradient (10 ml of 15–60% sucrose in 10 mM Hepes-NaOH and 1 mM MgCl2) and centrifuged for 18 h at 100,000 g. 1-ml fractions were taken from the top of the gradient and analyzed either directly or after TCA precipitation by Western blotting using antibodies against GFP (Santa Cruz Biotechnology, Inc.), Vps23 (a gift from S. Emr), Mnn9 (a gift from Y. Noda, University of Tokyo, Tokyo, Japan), and Kar2 (a gift from M. Rose, Princeton University, Princeton, NJ). Statistical analysis of the signal in each fraction was performed by quantifying the intensity of three independent experiments with the Odyssey 2.1 software (LI-COR Biosciences), and the percentages were plotted using Prism.
Online supplemental material