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.

Ubiquitination and cell surface delivery of Pma1-7 in gga1 gga2 cells. HA-Pma1-7 was induced for 2 h at 37°C in gga1 gga2 (CMY119) cells. (A) Localization of newly synthesized HA-Pma1-7. Cells were pulse labeled at 37°C, chased for 1 h, and fractionated on Renografin density gradients. HA-Pma1-7 was immunoprecipitated from each fraction and analyzed by SDS-PAGE and fluorography. Gas1 localization in each fraction was assayed by Western blot. (B) HA-Pma1-7 is ubiquitinated in gga1 gga2 but not sec18 (MPY36). HA-Pma1-7 was immunoprecipitated and then analyzed by Western blot with antiubiquitin followed by anti-HA. (C) gga2 is a suppressor of pma1-7. gga1 gga2 pma1-7 triple mutant (MPY33) was transformed with URA3-marked centromeric vectors bearing no insert or GGA2 (pCM53-3), gga2-ΔGAE (pCM62-2), gga2-Δhinge/GAE (pCM63-1), and gga2-ΔVHS (pCM64-1). Cells were spotted in serial 10-fold dilutions on plates with synthetic complete medium without uracil and incubated at 30 or 37°C.

Model for ubiquitination and intracellular transport of Pma1-7. Pma1-7 acquires ubiquitination (in grey) after ER export and before entry into the endosomal pathway. In mutants blocked in various steps of the endosomal pathway (vps8, vps45, pep12) before deubiquitination at the vacuole, Pma1-7 ubiquitination is increased. In the absence of ubiquitination in rsp5-1 and bul1 bul2, Pma1-7 (in black) is delivered to the plasma membrane. In vps1 and gga1 gga2 cells, ubiquitinated Pma1-7 is diverted to the plasma membrane where it undergoes deubiquitination (in black).

Pma1-7 is ubiquitinated. Cells bearing pMET-HA-pma1-7 (pMP4 or pS3) or pMET-HA-PMA1 (pWQ13) were induced for 2 h at 30 or 37°C to express tagged Pma1. (A) Pma1-7 induction in RSP5⁺ and rsp5-1 cells. Lysate was analyzed by anti-HA Western before (“0”) and after 2-h induction at 37°C (“on”). (B) Western blot with anti-ubiquitin (top) followed by anti-HA (bottom). HA-Pma1-7 was immunoprecipitated with anti-HA in bul1 bul2 (YHY009K), rsp5-1 (FW1810), tul1 (BY4742 background), vps45 (T. Stevens collection), vps4 (SEY6211 background), vps8 (WLX16-1A), pep12 (WLY74), and a variety of corresponding wild-type strain backgrounds. The position of Pma1 and the 116-kDa molecular weight marker are indicated by an arrow and dashed lines, respectively.

Pma1-7 fails to associate with rafts. Cells were shifted to 37°C for 2 h before lysis. After incubation with cold Triton X-100, samples were mixed with Optiprep, overlaid with an Optiprep step gradient, and centrifuged. Gradient fractions were analyzed after trichloroacetic acid precipitation. (A) Pma1 association with DIG-enriched complexes in wild-type and rsp5 (KY314) cells by Western blot with anti-Pma1. (B) DIG association of Pma1-7. Wild-type and rsp5 (KY314) cells bearing pMET-HA-pma1-7 (pMP4) were induced for 2 h at 37°C. Optiprep gradient fractions were analyzed by Western blot with anti-HA and anti-Gas1.

Ubiquitination of cell surface Pma1-7. Cells bearing pMET-HA-pma1-7 were induced to express HA-Pma1-7. (A) Newly synthesized Pma1-7 is stabilized in vps1. After induction for 1 h at 25°C, cells were shifted to 37°C and pulse labeled with Expre³⁵S³⁵S and chased for various times. HA-Pma1-7 was immunoprecipitated and analyzed by SDS-PAGE and fluorography. (B) Pma1-7 is routed to the plasma membrane in vps1. After pulse-labeling and chase for 1 h, cells were lysed and fractionated on Renografin density gradients. The distribution of HA-Pma1-7 on the gradient was determined by immunoprecipitation, whereas Gas1 and alkaline phosphatase were detected by Western blot. (C) Pma1-7 synthesis and degradation in VPS1 and vps1. HA-Pma1-7 level was determined by anti-HA Western blot before induction (“0”) and after 2-h induction at 37°C (“on”). Methionine (2 mM) was then added to terminate further synthesis of HA-Pma1-7 and incubation continued for another 2 h at 37°C (“off”). (D) Ubiquitination of HA-Pma1-7 in vps1 disappears after chase. vps1 (ACX58-3C), vps1 sec4 (MPY 39), sec4 (MPY35), and wild-type (L3852) cells were induced to express HA-Pma1-7 for 2 h at 37°C (on) and chased for another 2 h. HA-Pma1-7 was immunoprecipitated with anti-HA and analyzed by Western blot with anti-ubiquitin followed by anti-HA. Lane 1, Pma1-7 from isolated plasma membranes is not ubiquitinated. After induction of HA-Pma1-7 synthesis for 2 h followed by chase for another 2hat37°C, cell lysate was fractionated on Renografin density gradients in the presence of 5 mM NEM. Fractions 9 and 10 (arrow) containing peak distribution of HA-Pma1-7 (confirmed by Western blot with anti-HA, bottom) were collected for immunoprecipitation with anti-HA and analyzed by anti-ubiquitin Western blot.