Figure 3.

(A–D) Vps55p and Vps68p colocalize at endosomes and the vacuole-limiting membrane. The following strains were fixed and processed for double-label fluorescence microscopy. (A) Vps68-HA and Vps55-myc were coexpressed from plasmids in vps55 vps68 (BWY4) mutants and labeled with anti-myc mAb and anti-HA rabbit antiserum. (B) myc-tagged Vps21p was coexpressed with Vps68-HA from plasmids in vps68 cells (BY4741-6279) and double-labeled with anti-HA mAb and anti-myc rabbit antiserum. (C) Plasmid-expressed Vps68-HA was colocalized with Sec7-GFP in BWY10 cells labeled with rabbit anti-HA antiserum. (D) Plasmid-borne Vps68-HA was expressed in vps68 (BY4741-6279) cells double-labeled with an antiserum to endogenous Vph1 and anti-HA mAb. (E) Vps55p and Vps68p form a complex. Proteins were precipitated with anti-HA antiserum from extracts prepared from vps55 (BY4741-6842) cells expressing AU1-tagged Vps55, vps68 (BY4741-6279) cells expressing HA-tagged Vps68p, or vps55 vps68 (BWY4) cells expressing both tagged proteins, as indicated, and resolved by SDS-PAGE. Lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-AU1 mAbs. (F) Vps55p and Vps68p stabilize each other. vps55 (BY4741-6842; lane 1), vps68 (BY4741-6279; lane 4), or vps55 vps68 (BWY4; lanes 2, 3, and 5) strains containing plasmids for the expression of HA-tagged Vps55p and/or HA-tagged Vps68p were analyzed by Western blotting with anti-HA mAbs. Presence or absence of endogenous, untagged Vps68p or Vps55p is indicated. (G) The Vps55/68 complex contains a single copy of Vps55p. Detergent extracts were prepared from vps55 (BY4741-6842) mutant cells containing plasmids for the expression of AU1- and/or HA-tagged Vps55p (lanes 1–3 and 5–7), as indicated, and from vps55 vps68 (BWY4) expressing both AU1-tagged Vps55p and HA-tagged Vps68p (lanes 4 and 8). Proteins were immunoprecipitated with anti-HA antiserum and protein G-Sepharose, resolved by SDS-PAGE, and analyzed by Western blotting with anti-HA and anti-AU1 mAbs.