The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast.
Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.
Swaminathan
S
S
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
Amerik
A Y
AY
Hochstrasser
M
M
D002352:Carrier Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins; D010450:Endopeptidases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D006706:Homeostasis; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism* / Q000494:pharmacology; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C084434:DOA4 protein, S cerevisiae; C091908:END3 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C119627:VPS27 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease