FIGURE 8.
Bsd2 PY motifs are required to enhance WW-domain mediated Cps1 ubiquitination and sorting. A, Bsd2-sensitive cargo GFP-Cps1E7R was visualized by fluorescence microscopy in the bsd2Δ background co-expressing wild type or two different PY mutant forms of Bsd2. B, pep12Δ or pep12Δ bsd2Δ strains were transformed with vector alone or plasmids expressing the indicated forms of Bsd2 under control of the BSD2 promoter. Endogenous Cps1 was immunoprecipitated using anti-Cps1 polyclonal antibody and de-glycosylated to improve detection of ubiquitinated species. Detection of ubiquitin-Cps1 was by anti-ubiquitin monoclonal antibody. The same material was detected by anti-Cps1 polyclonal antibody as a loading control. C, an rsp5Δ strain expressing Rsp5 in which all three WW domains had been mutated was transformed with the indicated MVB cargo and visualized by fluorescence microscopy.