Figure 3.

Cross-linking radiolabeled assembly factor proteins and the 100-kD V-ATPase subunit. Wild-type cells were labeled with [³⁵S]methionine for 30 min followed by an equivalent 5-min pulse of label. Cells were converted to spheroplasts, osmotically lysed, and then treated with or without 250 μM DSP at room temperature for 30 min. The cross-linker was quenched with the addition of 50 mM Tris, pH 7.5, and cross-linked proteins were TCA precipitated, acetone washed, and then resuspended in denaturing buffer without reducing agent. Proteins were immunoprecipitated with anti-Vma22p polyclonal sera or preimmune sera and resuspended in buffer plus 5% β-mercaptoethanol and reimmunoprecipitated using anti-Vma22p, anti-Vph1p, anti-Vma12p, or anti-ALP polyclonal sera. Proteins from the second immunoprecipitation were resuspended in sample buffer, separated by SDS-PAGE, and exposed to a phosphor screen.