Figure 3.

Vac1p interacts with Vps45p and Vps21p in vivo. (A) Spheroplasts (5 OD₆₀₀ equivalents) of the following strains were prepared: GTY107 (Δvps45Δvac1) expressing Vac1HAp (lane 1), GTY104 (Δvac1) expressing Vac1HAp (lanes 2 and 3), and GTY104 expressing Vac1HAp and overexpressing Vps45p (lane 4). The spheroplasts were lysed, and the lysates were treated with (+) or without (−) the reducible cross-linker DSP (XL) as described. Vps45p and cross-linked proteins were immunoprecipitated with Vps45p antibodies. The immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose, and the blots were probed with HA antiserum. Vac1HAp was visualized using Blaze chemilluminescent detection reagents. (B) Spheroplasts of GTY112 (Δvac1Δvps21) (25 OD₆₀₀ equivalents) overexpressing Q66L-Vps21p (lane 1), Vps21p and Vac1HAp (lane 2), or Q66L-Vps21p and Vac1HAp (lane 3) were lysed and centrifuged at 16,000 × g. HA antisera (10 μl) was added to the cleared supernatants and incubated for 30 min at 4°C. Protein G-Sepharose was added, and the reactions were incubated an additional 30 min at 4°C. Sepharose–antibody–antigen complexes were pelleted at 2000 × g and washed five times with native lysis buffer. Proteins were eluted with sample buffer, resolved by SDS-PAGE, and subjected to Western analysis using Vps21p antisera. Prenylated (22 kDa, Vps21p) and unprenylated (23 kDa, Vps21p*) Vps21p were detected using Blaze chemilluminescent detection reagents.