FIG. 1.

Nhp6 protein physically associates with the CP complex. (A) Material immunoprecipitated by anti-Cdc68 antibody (P) from a whole-cell extract (strain W303-1a) and that remaining in the supernatant (S) were resolved electrophoretically on a 15% polyacrylamide–sodium dodecyl sulfate gel and analyzed by Western blotting with anti-Nhp6a antibody. The buffer for extract preparation and immunoprecipitation contained 350 mM NaCl, 50 mM potassium acetate, or 100 mM potassium acetate as indicated. Recombinant Nhp6a protein (25 ng) served as the marker. (B) Material immunoprecipitated (P) or remaining in the supernatant (S) after treatment of whole-cell extracts of cells containing HA-tagged Cdc68 and Pob3 proteins (strain NQ6811) with anti-HA monoclonal antibody was resolved (8 and 15% gels) and analyzed by Western blotting with anti-HA and anti-Nhp6a antibodies. The negative control (designated C) comprised a whole-cell extract of untagged cells (strain W303-1a) treated as described above. Immunoprecipitations are shown for two independent experiments. (C) Nhp6a and the CP complex associate in vitro. Nhp6a-V5-His₆ protein and CP containing Pob3-V5-His₆ were separately purified (from strains RJY6249 harboring p314NV5 and NB6244) using metal affinity resin as described in the text, mixed, and immunoprecipitated with anti-Cdc68 antibody. Resulting Western blots of purified and immunoprecipitated material were probed with anti-V5 antibody. Data are from two independent experiments. (D) Nhp6-associated CP complex associates with high-molecular-weight material. A whole-cell extract from cells containing Nhp6a-V5-His₆ protein (strain NB6241) made in 50 mM potassium acetate buffer was fractionated by centrifugation (1.5 h) at 200,000 × g to yield supernatant (S) and pellet (P) fractions. The pellet fraction was then resuspended in an original volume of 50 mM potassium acetate or 350 mM NaCl extraction buffer (P∗). Equal volumes were resolved (6-10-15% gel) and assayed by Western blotting with anti-Cdc68 or anti-V5 antibody. (E) Material immunoprecipitated from equal volumes of the indicated fractions with anti-Cdc68 or anti-V5 antibody was resolved (6-10-15% gels) and assayed by Western blotting using anti-Cdc68 and anti-V5 antibody.