Previous
experiments suggested that trafficking of the a-factor transporter
Ste6 to the yeast vacuole is regulated by ubiquitination. To define
the ubiquitination-dependent step in the trafficking pathway, we
examined the intracellular localization of Ste6 in the
deubiquitination-deficient doa4 mutant by immunofluorescence
experiments, with a Ste6-GFP fusion protein and by
sucrose-density-gradient fractionation. Loss of Doa4 function results
in a depletion of ubiquitin, particularly as the cells enter
stationary phase, presumably because ubiquitin gets degraded along
with the proteolytic substrate proteins. The doa4 mutation
should, therefore, interfere with all ubiquitin-dependent
processes. We found that Ste6 accumulated at the vacuolar membrane in
the doa4 mutant and not at the cell surface. Experiments with a
doa4 pep4 double mutant showed that Ste6 uptake into the lumen
of the vacuole is inhibited in the doa4 mutant. The uptake
defect could be suppressed by expression of additional ubiquitin
indicating that it is primarily the result of a lowered ubiquitin
level (and thus of reduced ubiquitination) and not the result of a
deubiquitination defect. Based on our findings, we propose that
ubiquitination is required for Ste6 sorting into the
multi-vesicular-bodies (MVB) pathway. In addition, we obtained
evidence suggesting that Ste6 recycles between an internal compartment
and the plasma membrane.
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