Calmodulin binding mutants of Rvs167 are defective in endocytosis
(A) Rvs167 mutants are defective in calmodulin binding in vivo. Lysates (1%) from wild-type (WT) or indicated Rvs167 mutant yeast cells expressing Cmd1-GFP (+) or untagged Cmd1 (−) were subjected to immunoprecipitation with anti-GFP antibodies. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by immunoblotting for Rvs167 or GFP. (B) Rvs167 mutants bind Rvs161. Lysates (1%) from WT or indicated Rvs167 mutant yeast cells were subjected to immunoprecipitation with anti-Rvs167 antibodies. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by immunoblotting for Rvs161 or Rvs167. As previously reported (Lombardi and Riezman, 2001), expression of Rvs161 is decreased in rvs167Δ cells. (C) Calmodulin binding mutants of Rvs167 are defective in LY endocytosis. The indicated strains were assessed for LY internalization by confocal microscopy. (D) Quantitation of vacuolar LY in strains presented in (C). Bars indicate the mean total vacuolar fluorescence per cell ± s.e.m. in arbitrary units (A.U.). **** p < 0.0001 compared to WT as determined by t-test. (E) Calmodulin overexpression partially rescues LY endocytosis in I138A and I142A mutant cells. The indicated strains expressing CMD1 from a multicopy vector (pRS426-CMD1) or vector alone (pRS426) were tested for LY uptake and the results quantified as in (D). **** p < 0.0001 as compared to empty vector as determined by t-test for each strain. Also see Figure S2. (F) Impaired Mup1-GFP endocytosis in Rvs167 mutants. Endocytosis of Mup1-GFP in the indicated strains was monitored by confocal microscopy 45 minutes after incubation in the presence (+Met) or absence of methionine (−Met). (G) Quantitation of Mup1-GFP internalization in strains presented in (F). Mup1-GFP internalization was quantified in the absence of methionine (closed bars) and 45 minutes after methionine addition (open bars) by determining the percentage of internalized Mup1-GFP signal as compared to total cell fluorescence. Bars show the mean ± s.e.m. of multiple independent transformants. **** p < 0.0001 compared to WT as determined by t-test.