Fig. 5.

The YKR020w gene product is a component of the VFT complex. A, Ykr020 binds Vps52, Vps53, and Vps54. Strains expressing Vps52-HA (PSY118), Vps53-HA (PSY119), or Vps54-HA (PSY120) and carrying a plasmid expressing either the PA-Ykr020 construct (pCuPAYKR020(416)) or PA alone (pRS416-CuProtA) were used to prepare detergent-solubilized extracts (Ext) as described under “Experimental Procedures.” IgG-Sepharose beads were used to affinity purify the PA fusions together with the associated proteins (IP). Eluted polypeptides were separated with SDS-PAGE and then visualized by immunoblotting with antiserum to HA. For each experiment 0.2% of the total lysate or 20% of the total eluate were loaded per gel lane. B, there is only one Ykr020 molecule per VFT complex. The experiment performed in panel A was repeated with a strain (PSY116) expressing Ykr020-Myc. C, Ykr020 is not required for the stability of Vps53. Wild type (WT, PSY119), vps52Δ (FRY116), and ykr020wΔ (FRY117) strains expressing Vps53-HA were grown to early log phase and proteins were precipitated with trichloroacetic acid. Proteins were separated by SDS-PAGE and analyzed by Western blot with anti-HA antibodies. Pgk1 was used to verify that the same amount of material was loaded on each gel lane. D, Vps52 is not necessary to maintain normal cellular levels of Ykr020. Wild type (WT, PSY116) and vps52Δ (FRY118) cells expressing Ykr020-Myc were analyzed as in panel C using anti-Myc antibodies.