Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway.
Degradation of various membrane proteins in the lumen of the vacuole/lysosome requires their prior sorting into the multivesicular body (MVB) pathway. In this process, ubiquitin serves as a sorting signal for most cargoes. The yeast ubiquitin hydrolase Doa4 acts late in the MVB pathway. It's role is to catalyze deubiquitination of cargo proteins prior to their sorting into the endosomal vesicles. This step rescues ubiquitin from degradation in the vacuole/lysosome, enabling it to be recycled. Accordingly, the level of monomeric ubiquitin is typically reduced in doa4 mutants. Although MVB sorting of cargo proteins is also impaired in doa4 mutants, the question of whether this defect is due solely to Doa4's role in maintaining a normal pool of ubiquitin in the cell remains open. We here show that the requirement of Doa4 for correct MVB sorting of the endocytic cargo general amino acid permease and of the biosynthetic cargo carboxypeptidase S are not because of the role of Doa4 in ubiquitin recycling. This suggests a direct role of Doa4 in MVB sorting and we show that this role depends on Doa4's catalytic activity. We propose that deubiquitination by Doa4 of cargo proteins and/or some components of the MVB sorting machinery is essential to correct sorting of cargoes into the MVB pathway.
Nikko
Elina
E
Laboratoire de la Physiologie Moléculaire de la Cellule, IBMM, Université Libre de Bruxelles, Rue des Professeurs Jeener et Brachet 12, B-6041 Gosselies, Belgium.
André
Bruno
B
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D004843:Epistasis, Genetic; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D022161:Transport Vesicles / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D026905:Amino Acid Transport Systems; C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; C491145:GAP1 protein, S cerevisiae; D008565:Membrane Proteins; D009687:Nuclear Proteins; C081224:PEP12 protein, S cerevisiae; D050765:Qa-SNARE Proteins; D000644:Quaternary Ammonium Compounds; C083890:SNF7 protein, S cerevisiae; C425333:STP22 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C107706:VAM3 protein, S cerevisiae; C107528:VPS4 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D002268:Carboxypeptidases; D010450:Endopeptidases; C019836:CPS1 protein, S cerevisiae; D043222:Ubiquitin Thiolesterase; C109028:UBP6 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D020559:Monomeric GTP-Binding Proteins; C499307:YPT6 protein, S cerevisiae