FIG. 4.

Expression, localization, and interaction between Cch1p and Mid1p are unaffected in pmr1 mutants. (A and B) Western blot analysis of epitope-tagged Cch1p and Mid1p variants was performed on total cell protein extracted from wild-type (WT), pmr1 mutant (pmr1), or untagged control (Δ) strains after growth in YPD medium to mid-log phase. Various amounts of each sample (either 1, 2, or 4 cell equivalents) were loaded and compared to endogenous cross-reacting proteins or mitochondrial porin as standards to control for slight variations in sample preparation or loading. (C) Immunofluorescence microscopy was performed on wild-type strains containing (top) or lacking (bottom) the epitope-tagged Cch1p-Myc variant. (D) Coimmunoprecipitation of Mid1p and Cch1p. Crude membranes were isolated from wild-type cells carrying Mid1p-HA (lanes 3 and 4) or Cch1p-Myc (lanes 2 and 4), solubilized in buffer containing Triton X-100, and immunoprecipitated using monoclonal antibodies to the HA epitope. Equal portions of the immunoprecipitated pellet and supernatant were then analyzed by Western blotting using either monoclonal antibodies to the Myc epitope (top) or polyclonal antibodies to the Pma1p protein (bottom). Cch1p but not Pma1p coprecipitated with Mid1p-HA (lane 4).