The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

Abstract

Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment. INTRODUCTIONMany cellular proteins are modified by the attachment of the 76-amino acid polypeptide ubiquitin (Hochstrasser, 1996;Hershko and Ciechanover, 1998). The main role of ubiquitination is to target proteins for degradation either directly by acting as a degradation signal that is recognized by the 26S proteasome or indirectly by sorting membrane proteins into the lysosomal/vacuolar degradation pathway (Hicke, 1999). Ubiquitination of substrate proteins, which occurs by a cascade of enzymatic reactions, is reversible. Ubiquitin can again be removed from proteins by deubiquitinating enzymes (Dubs). A large number of Dubs have been identified in various organisms that are either cysteine proteases or metalloproteases (Verma et al., 2002; Yao and Cohen, 2002). The more classical cysteine proteases can again be divided into two groups: the ubiquitin C-terminal hydrolases (Uchs) that preferentially cleave ubiquitin from peptides and small adducts (e.g., Yuh1 in yeast) and the extremely divergent family of ubiquitin-specific processing proteases (Ubps) that cleave ubiquitin from protein substrates (Hochstrasser, 1996). Of the 17 cysteine-protease-type Dubs in yeast 16 belong to the Ubp class.The degree of ubiquitination seems to be mainly regulated at the level of ubiquitin attachment. However, evidence is accumulating that deubiquitination also can be important for the regulation of ubiquitination levels. Evidence has been presented that the deubiquitinating enzyme Fat facets (Faf), which is involved in eye development in Drosophila, acts as a substrate-specific regulator of ubiquitination of the...

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