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 ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. 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 of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.
Losko
S
S
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Kopp
F
F
Kranz
A
A
Kölling
R
R
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D007425:Intracellular Membranes / Q000378:metabolism; D051938:Munc18 Proteins; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D018528:ATP-Binding Cassette Transporters; C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D006023:Glycoproteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497931:SEC1 protein, S cerevisiae; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase