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Fig. 3. Pma1-10 is stabilized in rsp5-1 cells. (A) Pulse-chase analysis in rsp5-1. Top panel, pma1-10 (XGY32) and PMA1⁺ (L3852) bearing pMET25-HA-pma1-10 (pKK4). Bottom panel, rsp5-1 (FY1810) and RSP5⁺ (FY354) bearing pKK7. Cells were exponentially growing at 25°C in minimal (top panel) or methionine- and cysteine-free synthetic complete medium (bottom panel), supplemented with 600 µM methionine. Cells were induced for 1 hour at 25°C, and then shifted to 37°C for 5 minutes before pulse-labeling for 5 minutes (top panel) or 10 minutes (bottom panel) with Expre³⁵S³⁵S. Cells were then chased for various times. Newly synthesized HA-Pma1-10 was immunoprecipitated and analyzed by SDS-PAGE and fluorography. (B) Ubiquitylation in rsp5-1. Cells were grown to mid-log phase in synthetic complete medium. Cells were induced to express HA-Pma1-10 for 2 hours at 25°C or 37°C. HA-Pma1-10 was immunoprecipitated with anti-HA antibody and analyzed by western blot with anti-ubiquitin. Arrow indicates position of Pma1 protein. The filter was then stripped and reprobed with anti-HA.

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