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. 2001 Jul 31;98(16):9104-9.
doi: 10.1073/pnas.161282998.

A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

X Gong  1 , A Chang
Affiliations

A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

X Gong et al. Proc Natl Acad Sci U S A. .

Abstract

Pma1 is a plasma membrane H(+)-ATPase whose activity at the cell surface is essential for cell viability. In this paper we describe a temperature-sensitive pma1 allele, pma1-10 (with two point mutations in the first cytoplasmic loop of Pma1), in which the newly synthesized mutant protein fails to remain stable at the cell surface at 37 degrees C. Instead, Pma1-10 appears to undergo internalization for vacuolar degradation in a manner dependent on End4, Vps27, Doa4, and Pep4. By contrast with wild-type Pma1, mutant Pma1-10 is hypophosphorylated and fails to associate with a Triton-insoluble fraction at 37 degrees C, suggesting failure to enter lipid rafts. Kinetic analysis reveals that, at the permissive temperature, newly synthesized Pma1-10 acquires Triton-insolubility before becoming stabilized. We suggest that phosphorylation and lipid raft association may play important roles in maintaining protein stability at the plasma membrane.

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Figures

Figure 1
Figure 1
Newly synthesized Pma1-10 is degraded in a Pep4-dependent manner. (A) Growth of cells on plates containing synthetic complete medium at 30°C and 37°C. Strains shown are wild-type (L3852), pma1-10 (XGY32), pma1-10 pep4Δ (XGX28–1A), and pma1-10 vps27Δ (XGX47–1D). (B) Pulse–chase analysis of Pma1-10. Cells were shifted to 37°C, labeled for 5 min with Expre35S35S, and chased for various times. Pma1 immunoprecipitation from cell lysate was normalized to acid-precipitable radiolabeled material, and analyzed by SDS/PAGE and fluorography. Strains analyzed are XGX28–1D, XGX44–1C, XGX28–1A, XGX44–1A, and XGX42–8B.
Figure 2
Figure 2
Cell surface delivery of Pma1-10. (A) Density gradient fractionation. Cells (XGY32) were pulse-labeled for 5 min and chased for 30 min at 24°C or 37°C. Lysates were prepared and separated on Renografin density gradients as described in Materials and Methods. Pma1 was immunoprecipitated from gradient fractions. Distribution of ALP and Gas1 was determined by Western blotting gradient fractions. (B) Specificity of immunoprecipitation. Anti-Pma1 antibody was incubated in the presence (+) or absence (−) of 5 μg of partially purified Neurospora Pma1 (gift of Carolyn Slayman, Yale University) before adding to RIPA buffer containing peak plasma membrane fractions (9 and 10) from pulse–chase experiments in A. Arrow indicates 50-kDa fragment. (C) Limited trypsinolysis. Total membranes were prepared from cells pulse-labeled for 2 min at 37°C and incubated in the absence (−) and presence of trypsin at 30°C for 5 and 15 min, as described in Materials and Methods. Immunoprecipitated Pma1 was analyzed by SDS/PAGE and fluorography.
Figure 3
Figure 3
Indirect immunofluorescence localization of Pma1-10. Cells were shifted to 37°C for 3 h, and then fixed, permeabilized, and stained with polyclonal anti-Pma1 and monoclonal anti-V-ATPase antibodies. (A) Pma1 localization in wild-type (L3852), pma1-10 end4–1 (XGX42–8B), pma1-10 (XGX28–1C), and pma1-10 pep4Δ (XGX28–1A) cells. Colocalization of Pma1 and V-ATPase at the vacuolar membrane is seen in pma1-10 pep4 cells. (B) Double labeling with anti-Pma1 and anti-V-ATPase in pma1-10 vps27Δ (XGX47–1D). Pma1 colocalizes in a large punctate spot with V-ATPase.
Figure 4
Figure 4
Degradation of Pma1-10 depends on Doa4. (A) Pulse–chase analysis of Pma1-10. pma1-10 doa4Δ cells (XGX55–5C) were shifted to 37°C, pulse-labeled for 5 min with Expre35S35S, and chased for various times. Pma1 and CPY were immunoprecipitated and analyzed by SDS/PAGE and fluorography. Arrow indicates mature CPY. (B) Indirect immunofluorescence localization of Pma1 in pma1-10 doa4Δ cells. Cells were grown at 25°C or shifted to 37°C for 3 h, and fixed, permeabilized, and stained with anti-Pma1 antibody followed by a Cy3-conjugated secondary antibody. Cells were also stained with DAPI, 4′,6-diamidino-2-phenylindole, to reveal nuclear localization.
Figure 5
Figure 5
At 37°C, Pma1-10 does not associate with a Triton-insoluble fraction. Wild-type (XGX28–1D) and pma1-10 pep4Δ (XGX28–1A) cells were pulse-labeled for 5 min at 37°C with Expre35S35S and chased for various times. Lysates were prepared, extracted with ice-cold Triton X-100, and separated into Triton-soluble and Triton-insoluble fractions, as described in Materials and Methods. T, total lysate; S, Triton-soluble; P, Triton-insoluble. Pma1 was immunoprecipitated and analyzed by SDS/PAGE and fluorography.
Figure 6
Figure 6
Kinetics of stabilization and acquisition of Triton-insolubility. (A) pma1-10 (XGY32) and pma1-10 pep4Δ (XGX28–1A) cells were pulse-labeled for 5 min at 24°C with Expre35S35S and chased at 24°C. At various times of chase, aliquots were removed and either placed on ice with sodium azide or shifted to 37°C for an additional 90-min incubation. Lysates were prepared, and Pma1 was immunoprecipitated and analyzed by SDS/PAGE and fluorography. (B) Lysates from pma1-10 cells (XGY32) pulse-labeled and chased at 24°C were extracted with ice-cold Triton X-100 and separated into Triton-soluble and Triton-insoluble fractions. Pma1 was immunoprecipitated and analyzed by SDS/PAGE and phosphorimaging. Both gel and quantitation are shown. Newly synthesized Pma1-10 is predominantly found in a Triton-insoluble fraction by 15-min chase, whereas resistance to degradation at 37°C is achieved by 1- to 2-h chase.
Figure 7
Figure 7
Pma1-10 is hypophosphorylated. Cells were shifted to 37°C, pulse-labeled for 5 min, and chased for 2 h. Pma1 was immunoprecipitated from cell lysate. Immunoprecipitates were divided, incubated in the presence (+) or absence (−) of alkaline phosphatase, and analyzed by extended electrophoresis on an 8% polyacrylamide gel followed by fluorography.
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