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. 2003 Dec;2(6):1361–1375. doi: 10.1128/EC.2.6.1361-1375.2003

FIG. 6.

FIG. 6.

Expression of mutant Cdr1p-GFPs and ATP- and substrate analog-binding characteristics, localizations, and FACS analysis of mutant Cdr1p-GFPs. (a) Expression of Cdr1p-GFP and mutant Cdr1p-GFPs in S. cerevisiae. The PM proteins (20 μg) from AD1-8u− (lane 1), PSCDR1-GFP (lane 2), SS5G (F774A) (lane 3), and SS6G (ΔF774) (lane 4) cells were separated on an SDS-8% polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and incubated with mouse monoclonal anti-GFP antibody (diluted 1:1,000) (upper panel), rabbit polyclonal anti-Cdr1p antibody (diluted 1:500) (middle panel), and rabbit polyclonal anti-Pma1p antibody (diluted 1:10,000) (lower panel). Proteins were immunodetected as described in Materials and Methods. (b) Flow cytometry of S. cerevisiae SS5G (F774A) and SS6G (ΔF774) cells. The flow cytometry of the S. cerevisiae cells was performed as described in the legend to Fig. 1e. The histogram derived from the CellQuest program depicts fluorescence intensities for AD1-8u− (control) (purple), SS5G (F774A) (orange), SS6G (ΔF774) (blue), and PSCDR1-GFP (green) cells. (c) Confocal pictures of S. cerevisiae cells expressing GFP-tagged wild-type and mutant Cdr1ps SS5G (F774A) and SS6G (ΔF774). Cells were grown in SD-URA medium to late log phase. The cells were washed and resuspended in an appropriate volume of 50 mM HEPES (pH 7.0). The cells were directly viewed on a glass slide with a drop of antifade reagent to prevent photobleaching, with a 100× oil immersion objective on a Bio-Rad confocal microscope (MRC 1024). (d) [α-32P]8-azido-ATP labeling of AD1-8u− (control), wild-type, SS5G (F774A), and SS6G (ΔF774) mutant Cdr1p-GFPs. The PM proteins (15 μg/50 μl) were photoaffinity labeled with 10 μM [α-32P]8-azido-ATP (10 μCi/nmol) in the absence (−) or presence (+) of 10 mM ATP as described in Materials and Methods. (e) [125I]IAAP labeling of AD 1-8u− (control [C]), wild-type (Cdr1p-GFP), and mutant Cdr1p-GFP from SS5G (F774A) and SS6G (ΔF774). The PM proteins (15 μg) were labeled with 3 to 6 nM [125I]IAAP (2,200 Ci/mmol) and competed as described in Materials and Methods. A 100 μM concentration of nystatin as indicated (+) was added to compete IAAP binding. (f) [3H]azidopine labeling of AD1-8u− (control [C]) and wild-type (Cdr1p-GFP) cells and mutant Cdr1p-GFPs from strains SS5G (F774A) and SS6G (ΔF774). The PM proteins (30 μg) were labeled with [3H]azidopine as described in Materials and Methods in the presence (+) or absence (−) of 100 μM miconazole as indicated.