Expression of yeast Rabs.
Yeast Rabs (see Table S1 in the supplemental material) under the control of the endogenous promoter and terminator were tagged with yeast-enhanced GFP (GenBank accession number U73901) at the open reading frame (ORF) 5′ terminus by PCR and cloned into either centromeric single-copy or integrating plasmids. Constructs were expressed at wild-type levels either in haploid cells as the only source of the Rab protein or in homozygous diploid cells where both copies were disrupted. The exceptions to the use of endogenous promoters were the Rab proteins Ypt10p and Ypt11p, which could barely be detected at endogenous levels, and the genes encoding these proteinswere typically expressed under the control of PYOP1
). Diploids were isolated on selective medium and subsequently sporulated at 23°C. Functionality was tested either by determining the ability of the tagged protein to act as the only cellular source of an essential gene to rescue a temperature-sensitive allele (Ypt31p/Ypt32p) or by observing the rescue of the mutant phenotype (Ypt7p, Ypt6p, and Ypt51p). YPT10
constructs under the control of Cu2+
were created using the CUP1-1
/YHR053C promoter, consisting of 330 bases from the start of CUP1
, and the endogenous YPT10
3′ region. The localization was monitored in haploid and diploid yeast cells (Novick laboratory or SC288C strain background); see Tables S2 and S3 in the supplemental material for yeast genotypes and plasmid constructs, respectively. Strains containing Sec61p-GFP and Spa2-GFP were constructed with the PCR-based integration system (37
). Yeasts were grown to log phase in sucrose-dextrose medium supplemented with amino acids as required at 25°C. GFP-Sec4p, GFP-Ypt31p, and GFP-Ypt32p were also cotransformed with a red fluorescence protein (RFP)-tagged nuclear protein to monitor the state of the nucleus during budding. Hoechst 33258 stain was used at 0.5 g/ml and incubated with the growing yeast in minimal media for 5 min followed by washing with growth media before mounting.
Transfection into mammalian cells.
HeLa cells (American Type Culture Collection, Rockville, MD) were maintained in alpha minimal essential medium containing 10% fetal calf serum, 100 U/ml penicillin, and 10 μg/ml streptomycin and passaged twice weekly. BHK cells (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 U/ml penicillin, and 10 μg/ml streptomycin and passaged twice weekly.
HeLa and BHK cells were transfected either with an Effectene transfection kit (QIAGEN) or with Lipofectamine 2000 (Invitrogen) according to the manufacturers' protocols. For transfection, cells were grown on 12-mm no. 1 coverslips in 24-well plates for approximately 24 h and transfected with 0.8 to 1 μg DNA. Transfections were typically allowed to proceed for 5 to 12 h before fixation and analysis.
Fluorescence microscopy procedures.
For live-cell microscopy of GFP-expressing yeast cells, the cells were grown to log phase, 2-μl aliquots were removed, and the cells were placed onto microscope slides under a no. 1 coverslip and observed immediately. Images were captured using a Nikon E600 microscope with a 60× objective (numerical aperture [NA], 1.4) and 2× Optivar or a 100× objective and 1× Optivar and an RT monochrome spot camera (Diagnostic Instruments, Inc., Sterling Heights, MI) driven by QED Image (QED Imaging, Inc., Pittsburgh, PA) or a Sensicam EM camera (Cooke Instruments, Inc.) driven with IPLab (Scanalytics). A Nikon remote focus accessory was used to capture stacks (0.2-μm slice size for yeast, 0.25 μm for mammalian cells) for deconvolution. Three-dimensional blind deconvolution was performed with AutoDeblur, version 9.1 (AutoQuant Imaging, Inc., Watervliet, NY). Stacks were deconvolved with 40 iterations using a medium- or low-noise correction level at the highest quality setting. All two-color images were first deconvolved in monochrome and then colored after deconvolution. Figures were made in Photoshop 7.0 (Adobe Systems, San Jose, CA). For wide-field microscopy of mammalian cells, cells were viewed on a Nikon Eclipse E600 fluorescence microscope using a 60× Plan Apo objective (NA, 1.4). Confocal microscopy was performed using an Olympus FluoView confocal station. Alexa 488 was excited with the 488-nm line of an argon laser, and Alexa 568 was excited with the 568-nm line of a krypton laser. Cells were viewed with a 60× Plan Apo objective lens (NA, 1.4), and images were captured with FluoView software (Olympus, Melville, NY). Endoplasmic reticulum (ER) inheritance assays were performed with an Olympus BX50 fluorescence microscope (100× objective; NA, 1.35) and TILLvisION software (TILL Photonics, Martinsried, Germany).
Indirect immunofluorescence microscopy.
Yeast cells in early log phase were immediately fixed in 3.7% formaldehyde for 20 min and fix replaced for 1 h. Cells were resuspended in spheroplasting buffer (100 mM KPi, pH 7.5, 1.2 M sorbitol), and 40 μg/ml Zymolase 20T was added. Cells were spheroplasted for 40 min at 37°C and allowed to settle onto polylysine-coated glass slides. Cells were permeabilized in 0.1% Triton X-100 and blocked in phosphate-buffered saline (PBS)-0.1% bovine serum albumin. The secondary antibodies used were Alexa 568-labeled goat anti-mouse immunoglobulin G (IgG) (Molecular Probes). The yeast endoplasmic reticulum was identified by using a monoclonal antibody against Pdi1p (EnCor Biotechnology). Nuclei were counterstained with Hoechst 33258 (5 μg/ml), and cells were mounted in ProLong Gold (Molecular Probes).
The preparation of HeLa cells for immunofluorescence microscopy was performed as described previously (63
). Briefly, cells were either fixed with 3% paraformaldehyde in PBS for 15 min, quenched with 50 mM NH4
Cl-PBS, and permeabilized for 5 min with 0.1% Triton X-100-PBS or fixed and permeabilized in cold methanol for 5 min. After blocking in 10% goat serum, cells were incubated with primary and secondary antibodies for 30 min each and mounted in Mowiol. The secondary antibodies used were Alexa 568-labeled goat anti-mouse IgG (Molecular Probes).
Antibodies and colocalization studies.
Early endosomes were localized using a monoclonal antibody directed against early endosome antigen 1 (EEA1) (Transduction Laboratories), late endosomes were localized using a monoclonal antibody directed against cation-independent mannose 6-phosphate receptor (Affinity BioReagents), lysosomes were localized using a monoclonal antibody directed against LAMP-1 (University of Iowa Hybridoma Bank), Golgi membranes were localized using a monoclonal antibody directed against the Golgi matrix protein GM130 (Transduction Laboratories), the trans-Golgi network was localized using a monoclonal antibody directed against TGN38 (Transduction Laboratories), and the endoplasmic reticulum was localized using monoclonal antibodies directed against protein disulfide isomerase (PDI) (Transduction Laboratories) or using ER-Tracker Blue-White DPX (Molecular Probes) at a concentration of 500 nM for 30 min at 37°C. The ER-Golgi intermediate compartment was localized using monoclonal antibodies against the KDEL receptor (Stressgen). To identify recycling endosomes, transferrin uptake assays were performed using Alexa 594-labeled human transferrin (kindly provided by Colin Parrish, Cornell University). HeLa cells were serum starved for 30 min, incubated with 50 μg/ml Alexa 594 transferrin for 20 min at 4°C, washed, and transferred to 37°C for 15 min before fixation.
Monoclonal anti-Rab 11 antibody was obtained from BD Transduction Labs. Cells were stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (LC Laboratories) at a concentration of 0.1 μM for 30 min at 37°C. To label the actin cytoskeleton, cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then incubated with tetramethyl rhodamine isocyanate (TRITC)-phalloidin (Sigma) at a concentration of 10 μg/ml for 10 min at room temperature.
For the quinacrine uptake assay, yeast cells in early log phase were harvested and resuspended in 500 μl yeast extract-peptone-dextrose (YEPD)-PO4, pH 7.6, with 2 mM quinacrine dihydrochloride and incubated for 5 min at room temperature in the dark. Cells were washed twice with YEPD-PO4, pH 7.6, and viewed immediately with a fluorescein isothiocyanate filter set. To label vacuolar membranes, a 500-μl volume of cells grown to early log phase was incubated with 12 μg/ml FM4-64 (Molecular Probes) for 15 min. The cells were then washed twice with fresh medium, resuspended in 5 ml of YEPD, and incubated for 45 min with shaking. To visualize, 1 μl of cells was gently harvested by centrifugation, mounted for microscopy, and visualized with a rhodamine filter set.