The deubiquitinating enzyme Ubp1 affects sorting of the ATP-binding cassette-transporter Ste6 in the endocytic pathway.
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.
Schmitz
Carolin
C
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Kinner
Andrea
A
Kölling
Ralf
R
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D043402:Cathepsin A / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology; D004705:Endocytosis; D010450:Endopeptidases / Q000378:metabolism / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005838:Genotype; D006023:Glycoproteins / Q000378:metabolism*; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D006023:Glycoproteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D013395:Sucrose; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D043402:Cathepsin A; C059237:ubiquitin-Nalpha-protein hydrolase