Abstract #95B

A role for the yeast Ubp16 deubiquitylating enzyme in mitochondrial phosphatidylserine transport. Rohan Baker, Marco Girhard. John Curtin School Medical Res, Australian National University, Canberra, Australia.
   Conjugation of ubiquitin to proteins can either mark them for degradation by the 26S proteasome or regulate their trafficking in the cell, depending on the extent of ubiquitylation. Deubiquitylating enzymes (DUBs) can cleave ubiquitin from such conjugates and can thus regulate the consequences of ubiquitylation. Ubp16 is one of 16 DUBs of the UBP family in yeast S. cerevisiae and has been reported to be inserted into the mitochondrial outer membrane by virtue of a putative transmembrane domain at its N-terminus (Kinner and Kolling, FEBS Lett. 549:135-140, 2003). However its function at the mitochondria (and in the cell in general) is unknown. Efforts to express full-length recombinant Ubp16 failed to produce soluble protein, presumably because of the transmembrane domain. Deletion of the predicted transmembrane domain enabled production and purification of a soluble protein. However, no DUB activity could be detected against model ubiquitin fusion proteins. Intact mitochondria purified from ubp16 yeast have lower DUB activity than those from wild-type yeast. Studies are continuing to identify possible substrates and/or other factors required for activity. The ubp16 null mutant has no reported phenotype. However, its mitochondrial location, and a previous report of a ubiquitin ligase subunit Met30p being required for mitochondrial phosphatidyl serine transport (Schumacher M. et. al., J. Biol. Chem., 277:51033-51042, 2002), prompted us to look for synthetic phenotypes. Indeed, psd2 null yeast dependent on the mitochondrial pathway for phosphatydylserine/ phosphatydylethanolamine biosynthesis have a grow defect in the absence of phosphatydylethanolamine when UBP16 is deleted. Double ubp16 psd2 mutants have abnormally dense mitochondria, similar to those of met30 mutants. Thus a Met30p/Ubp16p cycle of ubiquitylation/deubiquitylation may be required for correct phosphatydylserine transport to the mitochondria.

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