Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Cdc50-ts proteins seem to be rapidly inactivated at a non-permissive temperature. (A) Growth profiles of the cdc50-ts mutants after a shift to 37ºC. Wild-type (YKT38) (triangles), cdc50-11 (YKT993) (diamonds), and cdc50-162 (YKT942) (squares) cells were grown to early logarithmic phase in YPDA medium at 25ºC, shifted to 37ºC, and the number of cells (cells/ml) was counted at the indicated time points. (B) Localization of Drs2p-EGFP in the cdc50-11 mutant after a shift to 37ºC. Wild-type (YKT768) and cdc50-11 (YKT994) cells containing the DRS2-EGFP construct in the genome were grown to early logarithmic phase in SD medium at 25ºC, shifted to 37ºC, and incubated for the indicated time at 37ºC. Bar, 5 μm.
• Supplemental Figure 2 - Kex2p-EGFP is localized to vacuoles in the cdc50-ts mutant. Wild-type (YKT903) and cdc50-11 (YKT1000) cells containing the KEX2-EGFP construct in the genome were grown for 3 h at 25ºC or 37ºC in SD medium, followed by staining with 100 μM CellTracker Blue CMAC at 25ºC for 15 min. Arrows indicate colocalization of Kex2p-EGFP with CellTracker Blue CMAC.
• Supplemental Figure 3 - The effects of overexpression of the GDP- or GTP-bound form of Ypt32p on the recycling of GFP-Snc1p. Wild-type (YKT38) cells harboring pRS416-GFP-SNC1 or pRS416-GFP-SNC1pm were transformed with pKT1578 (YEplac181-YPT32^Q72L; YPT32-GTP) or pKT1579 (YEplac181-YPT32^S27N; YPT32-GDP). Cells were grown to mid logarithmic phase at 30ºC in SD-Leu-Ura medium. Numbers indicate the percentages of cells in which GFP-Snc1p accumulated in large compartments located in the bud or near the bud neck. Bar, 5 μm.