title: Characterization of an M28 metalloprotease family member residing in the yeast vacuole.
abstract: The systematic and complete characterization of the Saccharomyces cerevisiae genome and proteome has been stalled in some cases by misannotated genes. One such gene is YBR074W, which was initially annotated as two independent open reading frames (ORFs). We now report on Ybr074, a metalloprotease family member that was initially predicted to reside in the endoplasmic reticulum (ER). Therefore, we tested the hypothesis that Ybr074 may be an ER quality control protease. Instead, indirect immunofluorescence images indicate that Ybr074 is a vacuolar protein, and by employing protease protection assays, we demonstrate that a conserved M28 metalloprotease domain is oriented within the lumen. Involvement of Ybr074 in ER protein quality control was ruled out by examining the stabilities of several well-characterized substrates in strains lacking Ybr074. Finally, using a proteomic approach, we show that disrupting Ybr074 function affects the levels of select factors implicated in vacuolar trafficking and osmoregulation. Together, our data indicate that Ybr074 is the only multispanning vacuolar membrane protease found in the yeast Saccharomyces cerevisiae.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Hecht
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Wytiaz
authors.forename: Tslil
authors.initials: T
authors.lastname: Ast
authors.forename: Maya
authors.initials: M
authors.lastname: Schuldiner
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
mesh_terms: D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000235:genetics; D020543:Proteome / Q000235:genetics* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology* / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D045726:Metalloproteases
keywords: Saccharomyces cerevisiae; integral membrane; protease