Figure 1.

Intracellular distribution of ubiquitinated Ste6. Whole cell extracts of the Δste6 strain RKY959 transformed with YEp112 (2μ-CUP1-UBI-HA) and pYKS2 (2μ-STE6-c-myc) were fractionated on a sucrose density gradient (20–50% [wt/wt] sucrose, fraction 1: low sucrose density). The gradient fractions were analyzed by Western blotting for the presence of marker proteins with specific antibodies against HA-tag (BAbCo) (A) , Ste6 (B), Pep12 (C), ALP (D; Molecular Probes, Eugene, OR), and Pma1 (E). In A, immunoprecipitates were analyzed for the presence of HA-tagged ubiquitin covalently linked to Ste6 after immunoprecipitation with anti-Ste6 antibodies.