QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02749

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, Y. Articles by Chang, A. Search for Related Content PubMed PubMed Citation Articles by Liu, Y. Articles by Chang, A.

Quality control of a mutant plasma membrane ATPase: ubiquitylation prevents cell-surface stability

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA

View larger version (52K): [in a new window]   Fig. 1. Pma1-10 is slow to exit the ER. Indirect immunofluorescence localization of Pma1. Synthesis of HA-tagged Pma1 was induced at 37°C for 2 hours (ON), and HA-Pma1-10 synthesis was then shut-off for an additional 2 hours, as described in the Materials and Methods. Cells were fixed and stained with anti-HA and anti-Kar2 before synthesis (0), after protein induction, and after shut-off. HA-tagged wild-type Pma1 was localized in wild-type cells (WLY104) and HA-Pma1-10 was localized in wild-type (KKY1) (A) and pep4 cells (KKY67) (B). Arrows indicate ER-localized rings. Images were captured at the same exposure time and adjusted with Adobe Photoshop software using the same settings.

View larger version (46K): [in a new window]   Fig. 2. Ubiquitylation of Pma1-10 at or before arrival at the plasma membrane is reversible. Induction and shut-off of synthesis of HA-tagged Pma1 was at 37°C. (A) Western blot with anti-HA antibody before HA-Pma1-10 synthesis (0), after induction for 1 hour (on) and chase for 2 hours (off) in end3-1 (KKY81) and END3 (KKY80) cells. (B) HA-tagged mutant was immunoprecipitated with anti-HA from end3-1 and END3 cells; wild-type HA-Pma1 was immunoprecipitated from END3 cells. Immunoprecipitates were blotted with anti-ubiquitin. Arrow indicates position of Pma1 protein. After stripping, the filters were blotted again with anti-HA.

View larger version (35K): [in a new window]   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.

View larger version (69K): [in a new window]   Fig. 4. Turnover of Pma1-10 requires the epsin UIM domain. (A) FM 4-64 endocytosis assay. ENT1+ [ent1 ent2 ede1 cells carrying pEDE1 and pENT1 (LHY3185)] and ent1UIM [ent1 ent2 ede1 cells carrying pent1uim (LHY3156)] cells were grown in YPD, labeled for 15 minutes at 30°C with 40 µM FM 4-64. Cells were then washed and resuspended in fresh YPD for 45 minutes at 30°C before visualization. (B) Pulse-chase analysis. ENT1+ (L3852) and ent1UIM cells growing exponentially in minimal medium were induced to express HA-Pma1-10 (pKK7) for 1 hour at 25°C. Cells were then shifted to 37°C for 5 minutes before pulse-labeling with Expre35S35S followed by chase for various times. Immunoprecipitations from lysate with anti-HA antibody were normalized to acid-precipitable cpm, and analyzed by SDS-PAGE and fluorography. (C) Indirect immunofluorescence localization of Pma1-10 in ent1UIM cells. Cells growing exponentially were induced to express HA-Pma1-10 (pKK42) for 1 hour at 37°C and then `chased' in the presence of 2 mM methionine for 2 hours at 37°C. Cells were fixed before induction (0), after induction (on), and after shut-off (off), and stained with anti-HA antibody followed by CY3-conjugated secondary antibody. Images were captured at the same exposure time and adjusted with Adobe Photoshop software using the same settings.

View larger version (80K): [in a new window]   Fig. 5. Retention of Pma1-10 at the cell surface in end3 cells results in increased phosphorylation and association with DIGs. (A) Association with Pma1-10 with detergent-resistant membranes. HA-Pma1-10 was induced and chased at 37°C in pep4 (KKY67), end3 (KKY81), and yvh1 (KKY66) cells. Lysate was incubated with 1% ice-cold Triton X-100 for 30 minutes on ice, and then placed at the bottom of an Optiprep gradient, as described in Materials and Methods. After centrifugation, six fractions were collected from the top of the gradient, proteins were acid-precipitated, and HA-Pma1 was analyzed by western blot. Pma1 and ALP were analyzed as Triton-insoluble and -soluble markers, respectively. (B) Localization of Pma1-10 in end-3-1 cells by cell fractionation on Renografin density gradients. HA-Pma1-10 was induced for 2 hours in end3-1 cells (KKY65) and then chased for 2 hours at 37°C. Lysate was then fractionated and HA-Pma1-10 localization was determined by western blot of membrane fractions. (C) Phosphorylation of Pma1-10. Electrophoretic mobility was analyzed after pulse-labeling and chase for 2 hours at 37°C by SDS-PAGE for an extended time. Strains are end4-1 (XGX42-8D), end4-1 pma1-10 (XGX42-8B) and pep4 pma1-10 (XGX28-1A).

View larger version (21K): [in a new window]   Fig. 6. Pma1-Ub can associate with detergent-resistant membranes. Wild-type cells (F1105=W303; MAT ura3-1 leu2-3,112 his3-11 trp1-1 ade2-1 can1-100) bearing pXZ28 and pLH609 were induced to express HA-tagged Pma1 or Pma1-Ub for 2 hours by shifting from raffinose medium to galactose-containing medium. Cell lysates were analyzed for detergent insolubility on Optiprep gradients. Pma1 was analyzed by western blot. Gas1 and ALP were analyzed as Triton-insoluble and soluble markers, respectively. Ubiquitylation of Pma1 does not cause its exclusion from detergent-resistant membranes.

View larger version (65K): [in a new window]   Fig. 7. yvh1 is a suppressor of pma1-10, causing cell-surface stability of Pma1-10. (A) Cells were grown at 30 and 37°C on plates with selective synthetic complete medium. (B) Pulse-chase analysis of Pma1-10 in YVH1 (XGY32) and yvh1 (KKY11) cells. Cells were pulse-labeled with Expre35S35S for 5 minutes and chased for various times. Immunoprecipitations with anti-Pma1 antibody were normalized to acid-precipitable cpm. (C) Endocytosis in yvh1 cells. Cells bearing pGAL-myc-STE3 (pSL2015) were grown in galactose-containing medium to induce expression of myc-Ste3. At various times after shifting to glucose, cells were lysed and myc-Ste3 was analyzed by western blot. (D) Pma1-10 is delivered to the plasma membrane in yvh1 cells. pma1-10 yvh1 (KKY11) and pma1-10 pep4 (XGX28-1A) cells were pulse-labeled for 5 minutes and chased for 1 hour at 37°C. Lysate was fractionated on Renografin density gradients. Newly synthesized Pma1-10 was immunoprecipitated from pooled fractions and analyzed by SDS-PAGE and autoradiography. Distribution of the cell-surface marker Gas1 was analyzed by western blotting of pelleted membranes. (E) Electrophoretic mobility of newly synthesized Pma1. Wild-type, yvh1 pma1-10, and pep4 pma1-10 cells were pulse-labeled and chased for 2 hours at 37°C. Pma1 was immunoprecipitated with anti-Pma1 antibody and analyzed by SDS-PAGE and autoradiography.

View larger version (59K): [in a new window]   Fig. 8. Suppression of Pma1-10 ubiquitylation independent of slow ER export. Cells bearing HA-tagged Pma1 were induced for 2 hours at 37°C and then chased for another 2 hours. (A) Indirect immunofluorescence localization of HA-Pma1-10 in yvh1 cells. Cells were fixed and stained with anti-HA and anti-Kar2, as described in Materials and Methods. Digital images were captured at the same exposure time and adjusted with Adobe Photoshop at the same settings. (B) Pma1-10 ubiquitylation, but not Pma1-7 ubiquitylation, is prevented in yvh1. Wild-type (L3852) and yvh1 (KKX15-1B) bearing pS3 (MET25-HA-pma1-7) or pKK5 (MET25-HA-pma1-10) were induced for 2 hours at 37°C. Anti-HA immunoprecipitates were analyzed by western blotting with anti-ubiquitin and anti-HA antibodies. Arrow indicates position of non-ubiquitylated Pma1.