FIGURE 6.

Bsd2 enhances Cps1 ubiquitination and MVB sorting. A, indicated full-length GFP-Cps1 (wild type and mutant) constructs were expressed in an isogenic background in which the BSD2 gene had been deleted. B, same GFP-Cps1 constructs were immunoprecipitated and visualized as in 5C from yeast lacking both PEP12 and BSD2 to assess the ability of Rsp5 to ubiquitinate these forms of Cps1 in the absence of Bsd2. C, maturation kinetics of wild type, E7R, or K8R forms of GFP-Cps1 were compared in the wild type background compared with the isogenic strain lacking BSD2 (bsd2Δ). Yeast cells were labeled with [³⁵S]cysteine and methionine. Following a chase with unlabeled amino acids, cell lysates were immunoprecipitated with polyclonal anti-Cps1 antibody. The GFP moiety is rapidly cleaved following delivery to the vacuolar lumen, therefore kinetics of delivery to the lumen of the vacuole were assessed by monitoring the disappearance of the full-length form of GFP-Cps1 at the indicated time points by phosphorimaging.