Figure 2.
Pma1-7 is routed to the plasma membrane in the absence of ubiquitination in rsp5 and bul1 bul2 cells. Cells were induced to express HA-Pma1-7 in the absence of methionine for 1 h at 25°C. Cells were shifted to 37°C for 15 min, pulse labeled 10 min with Expre35S35S, and chased for various times. (A) Stabilization of Pma1-7. Wild-type (L3852), rsp5-1 (FW1810), and bul1 bul2 (YHY009K) cells bearing MET-HA-pma1-7 (pMP4). HA-Pma1-7 was immunoprecipitated from cells lysed at various times of chase and analyzed by SDS-PAGE and fluorography. (B) Cell fractionation on Renografin density gradients. HA-Pma1-7 was induced in pep4 (ACY17) and bul1 bul2 cells followed by pulse labeling and chase for 1 h at 37°C. After gradient centrifugation, HA-Pma1-7 was immunoprecipitated from each fraction. Plasma membrane (Gas1) and vacuolar membrane (alkaline phosphatase, ALP) markers were assayed by Western blot after pelleting membranes from each fraction.