Topological organization of the Rieske iron-sulphur protein and subunit IV in the cytochrome bc1 complex of Rhodobacter sphaeroides.
The ubiquinol-cytochrome c2 oxidoreductases (cytochrome bc1 complex) of Rhodobacter sphaeroides contains highly conserved cytochrome b, cytochrome c1 and Rieske FeS subunits, as well as a unique 14 kDa polypeptide, designated as subunit IV, thought to function as a ubiquinol-binding protein [Yu and Yu (1991) Biochemistry 30, 4934-4939]. As the topology of subunit IV is unknown and that of the FeS subunit remains a matter of debate, both the inner (cytoplasmic) and outer (periplasmic) surfaces of the intracytoplasmic membrane (ICM) were digested with proteinase K, and cleavage products were identified by immunoblotting. In uniformly oriented chromatophore vesicles (inner ICM surface exposed), fragments of approx. 4 and 1 kDa were removed from subunit IV and the FeS protein respectively. Neither subunit IV nor the FeS protein was cleaved from the outer ICM surface as exposed in osmotically protected spheroplasts or as presented to proteinase K after microencapsulation of the protease in unilamellar liposomes and fusion of these structures to chromatophore vesicles. Studies with the isolated bc1 complex, however, suggested that the C-terminal domain of the Rieske FeS, thought to reside on the periplasmic side of the ICM, was resistant to proteinase K. Overall, these results suggest a single N-terminal transmembrane helix for the FeS protein, with exposure of the N-terminus to the cytoplasm and an orientation in which a major, N-terminal portion of subunit IV is located in the cytoplasm with the predicted C-terminal transmembrane domain anchoring this polypeptide to the membrane.
Wu
J
J
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855-1059.
Niederman
R A
RA
D020130:Bacterial Chromatophores / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002918:Chymotrypsin / Q000378:metabolism; D014450:Electron Transport Complex III / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000648:ultrastructure; D012697:Serine Endopeptidases / Q000378:metabolism; D013104:Spheroplasts / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D010446:Peptide Fragments; C037989:Rieske iron-sulfur protein; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D019286:Endopeptidase K; D014450:Electron Transport Complex III