PMC

Endogenous PHS levels are not significantly elevated in either the absence of Rsb1 or the absence of Pdr5 and Yor1. Wild-type, rsb1Δ, rsb1Δ dpl1Δ, dpl1Δ, pdr5Δ yor1, and pdr5Δ yor1 rsb1Δ strains were grown to mid-log phase and processed for HPLC analysis.

Interaction of Bul1 with Rsb1 and Pdr5/Yor1. Wild-type or isogenic mutants lacking the indicated genes were grown to mid-log phase and spotted onto YPD plates containing varying concentrations of PHS.

Tryptophan uptake is increased in the pdr5Δ yor1 strain and decreased in the rsb1Δ strain. A, wild-type (diamonds) and pdr5Δ yor1 (squares) strains were grown to mid-log phase and harvested. Cells were subjected to tryptophan uptake assays as described above. Time points were taken at 10-min intervals for 40 min. B, wild-type (diamonds) and rsb1Δ (squares) strains were grown and assayed as in A.

Tat2 is present at the plasma membrane longer in the absence of Pdr5 and Yor1. Wild-type and pdr5Δ yor1 strains were transformed with pRS316-Cup1-Tat2-GFP-3×HA (pSR74), and transformants were grown overnight in CSM −URA media. These cultures were shifted to CSM −URA media containing 4 μg/ml tryptophan, grown for 6 h, and visualized by Nomarski optics and fluorescence microscopy.

Bypass of rsb1Δ by pdr5Δ yor1. Wild-type and isogenic derivatives lacking the indicated genes were grown in YPD to mid-log phase and spotted onto YPD plates containing the indicated concentrations of PHS (A), HePC (B), SDS (C), or YPD alone. D, these same strains were spotted onto minimal media plates containing the indicated concentrations of tryptophan. Strains lacking Rsb1 are sensitive to all three compounds, whereas strains lacking both Pdr5 and Yor1 show increased resistance.

Tat2 stability correlates with PHS resistance. The indicated strains were transformed with either pRS316-CUP1-Tat2-GFP-3×HA (pSR74) or empty vector (pRS316). Transformants were grown to mid-log phase and labeled with [³⁵S]methionine for 10 min. A large excess of unlabeled methionine was added, and samples were withdrawn at the indicated times. Cells were lysed and processed for immunoprecipitation using an anti-HA antibody as described previously (). Immunoprecipitates were resolved on SDS-PAGE, and levels of Tat2-eGFP-HA were determined using a phosphorimager.

Multiple phenotypes are affected by tryptophan prototrophy. Wild-type cells transformed with either a low copy URA3 plasmid (pRS316) or a low copy TRP1 plasmid (pRS314) were grown in selective minimal media to mid-log phase and spotted onto YPD plates containing the indicated drug concentration or YPD alone. Trp⁺ (pRS314) strains were more resistant to PHS, SDS, and HePC as compared with their Trp⁻ (pRS316) counterparts, indicating that tryptophan status influences multiple phenotypes.

Bulk internalization is slowed in the pdr5Δ yor1 strain. A, wild-type and pdr5Δ yor1 strains were grown to mid-log phase and chilled on ice. FM4-64 dye was added to pre-chilled cells, and cells were incubated at 4 °C for 45 min. Samples were harvested and resuspended in pre-warmed YPD and incubated at 30 °C for the indicated time points. At the designated time points, samples were washed with cold SC-azide buffer and visualized by Nomarski optics (left panels) or fluorescence microscopy (right panels). B, quantification of the percentage of pixels (fluorescence) on the vacuolar membrane as compared with the whole cell was determined as described under “Experimental Procedures.”

C-terminal truncations of Rsb1 show differential expression and PHS tolerance. A, predicted topology of Rsb1. A prediction for the organization of Rsb1 made by the program RbDe is shown. The locations of previously determined N-linked glycosylation sites in the luminal N terminus () are graphically represented by the diagrams. The positions of the C-terminal truncation mutations are indicated by arrows. B, low copy plasmids containing the above forms of Rsb1, along with an empty vector control, were transformed into wild-type and rsb1Δ strains. These transformants were grown to mid-log phase in selective minimal media and spotted onto YPD plates containing varying concentrations of PHS as described above. C, these same cultures from A were extracted using the TWIRL method (as described under “Experimental Procedures”), and equal amounts of total protein were run on SDS-PAGE, transferred to nitrocellulose, and probed using an anti-HA epitope antibody. These membranes were stripped and re-probed for Pma1.