The yeast deubiquitinating enzyme Ubp16 is anchored to the outer mitochondrial membrane

Abstract

We looked for membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitinspeci¢c processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at membranes. For each of the Ubps examined, a certain fraction was found to be membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the membrane fraction suggesting that this protein performs a major function at membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively membrane associated. By cell fractionation and immuno£uorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast outer mitochondrial membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral membrane protein of the outer mitochondrial membrane with an N in^Cout orientation. Phenotypic characterization of the v vubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria. ß

Proteomic Analysis of the Yeast Mitochondrial Outer Membrane Reveals Accumulation of a Subclass of Preproteins

“…We showed for Rho1, another member of the Rho family identified in the outer membrane proteome, that the in vitrosynthesized protein specifically associates with mitochondria. Another interesting group is formed by proteins involved in ubiquitin-dependent protein degradation, including a ubiquitin-regulatory protein (Sel1) and the ubiquitin-specific protease Ubp16, which was recently found to reside in the mitochondrial outer membrane (Kinner and Kö lling, 2003). Two of the new proteins, Ykl027w and Yhr003c, contain motifs that are found in ubiquitin-activating enzymes.…”

Nonredundant Roles of Mitochondria-associated F-Box Proteins Mfb1 and Mdm30 in Maintenance of Mitochondrial Morphology in Yeast

“…Consistent with a specific role of ubiquitylation of mitochondrial proteins, the mitochondrial outer membrane harbors a deubiquitinating enzyme, Ubp16. However, neither deletion nor overexpression of the UBP16 gene produces any obvious phenotype, and mitochondrial morphology and inheritance are not affected in ubp16 mutants (Kinner and Kö lling, 2003). What might be the role of Mfb1 and Mdm30 in mediating turnover of Fzo1 and other mitochondrial target proteins?…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…Other DUBs seem to function specifically on endosomes and multivesicular bodies, such as Doa4/Ubp4 (Luhtala and Odorizzi 2004;Amerik et al 2006). One DUB, Ubp16, is thought to be an integral membrane protein and fractionates with mitochondria (Kinner and Kölling 2003). The enzymatic specificity of DUBs from yeast is only partially characterized (Amerik et al 2000b;Schaefer and Morgan 2011).…”