Evidence for a Direct Role of the Doa4 Deubiquitinating Enzyme in Protein Sorting into the MVB Pathway

• Published in: Traffic (Copenhagen, Denmark) Vol. 8; no. 5; pp. 566 - 581
• Main Authors: Nikko, Elina, André, Bruno
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2007
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Transport Systems - genetics; Amino Acid Transport Systems - metabolism; Carboxypeptidases - genetics; Carboxypeptidases - metabolism; Cell Membrane - metabolism; CPS; Cytosol - metabolism; degradation; endocytosis; Endocytosis - drug effects; Endocytosis - physiology; Endopeptidases - genetics; Endopeptidases - metabolism; Endopeptidases - physiology; Endosomal Sorting Complexes Required for Transport; Epistasis, Genetic; Gap1; Immunoblotting; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microscopy, Fluorescence; Models, Biological; Monomeric GTP-Binding Proteins - genetics; Monomeric GTP-Binding Proteins - metabolism; Mutation; MVB sorting; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Protein Transport - drug effects; Protein Transport - physiology; Qa-SNARE Proteins - genetics; Qa-SNARE Proteins - metabolism; Quaternary Ammonium Compounds - pharmacology; recycling; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Transport Vesicles - metabolism; ubiquitin; Ubiquitin - genetics; Ubiquitin - metabolism; ubiquitin hydrolase; Ubiquitin Thiolesterase; vacuole; Vacuoles - metabolism; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 1398-9219; 1600-0854
• DOI: 10.1111/j.1600-0854.2007.00553.x

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 doa4mutants. Although MVB sorting of cargo proteins is also impaired in doa4mutants, 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.