Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 mutant

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiq uitination. To define the ubiquitinationdependent step in the trafficking p athway, we examined the intracellular localization of Ste6 in the ubiquitin ation-deficient doa4 mutant by immunofluorescence experiments, with a Ste6- green fluorescent protein fusion protein and by sucrose density gradient fr actionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 doub le mutant showed that Ste6 uptake into the lumen of the vacuole is inhibite d in the doa4 mutant. The uptake defect could be suppressed by expression o f additional ubiquitin, indicating that it is primarily the result of a low ered ubiquitin level (and thus of reduced ubiquitination) and not the resul t of a deubiquitination defect. Based on our findings, we propose that ubiq uitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence s uggesting that Ste6 recycles between an internal compartment and the plasma membrane.