Membrane topography of the subunits of ubiquinol – cytochrome‐c oxidoreductase of Saccharomyces cerevisiae

Abstract

The topology of the subunits of ubiquinol–cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae has been determined using a digitonin/proteinase K assay. With this assay we were able selectively to disrupt the mitochondrial membranes and to identify the subunits which became proteinase‐K‐sensitive after disruption of either the outer or both outer and inner membranes. This approach confirmed previous indications for the localization of the core I protein, cytochrome c1, cytochrome b, the FeS protein and the 17‐kDa subunit, while it also provided direct evidence for the site of accessibility to proteinase K of the 14‐kDa and 11‐kDa subunits. The 14‐kDa subunit faces the mitochondrial matrix and the 11‐kDa subunit faces the intermembrane space.

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

“…Triton X-100 solubilizes all membranes and therefore makes all proteins accessible for proteinase K. Panel C shows results obtained for mitochondrial proteins facing either the matrix (FI~) or the IMS (ISP42). Panel A shows the localisation of subunits VII and VIII as was determined before [9]. Resistance to proteinase K of the 14-kDa subunit VII (lane 5) shows that this protein is not exposed to the IMS, consistent with earlier findings which indicated that this protein faces the mitochondrial matrix [9].…”

“…This subunit was shown to be located at the matrix side of the inner membrane [9]. In yeast this pro- Mitochondria were treated as described in Section 2.…”

“…Antibodies against the c-myc epitope were obtained from Cambridge Research Biochemicals and antibodies against the HA epitope from Boehringer-Mannheim. The antisera used to detect subunits VII and VIII are described in [9]. Mitoplasts were obtained by diluting the mitochondrial suspension 3-fold in 20 mM Tris-HC1 pH 7.4, incubating the suspension 15 min on ice, sedimenting the mitoplasts by centrifugation and resuspending them in 0.6 M sorbitol, 20 mM KPi pH 7.5, 1 mM EDTA, 1 mM MgC12.…”

“…A similar behaviour was displayed by another low molecular weight protein of the complex, subunit VII, which faces the matrix side. A model for the topology of these subunits in the membrane is discussed with respect to the structure of the complex and their involvement in quinone binding.,~?ey words: Membrane topology; Ubiquinol-cytochrome c ~.xidoreductase; Epitope tagging; Integral membrane protein; ( ~accharomyces cerevisiae) tein [8], have shown that at least this part of subunit VIII is exposed to the intermembrane space [9]. In addition, hydrophobicity plots of subunit VIII from S. cerevisiae, K. lactis, S. pombe and the homologous subunit from bovine heart predict the presence of a conserved hydrophobic stretch potentially capable of spanning a lipid bilayer (Fig.…”

“…,~?ey words: Membrane topology; Ubiquinol-cytochrome c ~.xidoreductase; Epitope tagging; Integral membrane protein; ( ~accharomyces cerevisiae) tein [8], have shown that at least this part of subunit VIII is exposed to the intermembrane space [9]. In addition, hydrophobicity plots of subunit VIII from S. cerevisiae, K. lactis, S. pombe and the homologous subunit from bovine heart predict the presence of a conserved hydrophobic stretch potentially capable of spanning a lipid bilayer ( Fig.…”