Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein.
The mode of membrane attachment of five subunits from Chlamydomonas reinhardtii cytochrome b6f complex has been studied using biochemical approaches. Antisera specific for cytochrome f, cytochrome b6, the Rieske iron-sulfur protein, subunit IV, and a 4-kDa subunit (product of the petG gene) were used to quantify the degree of extraction of each of these polypeptides following various treatments. In contrast to the other four subunits, the Rieske protein was extracted to extents varying between 50 and 100% following two cycles of freezing and thawing in the presence of chaotropic agents (KSCN, urea, or NaI). The Rieske protein was not extracted by 2 M NaCl and was rather resistant to alkaline treatments, being extracted by 20 mM 3-(cyclohexylamino)propanesulfonic acid buffer only at pH > 11.5. The hydrodynamic behavior of the isolated Rieske protein was examined in the absence and presence of detergent by ultracentrifugation and by molecular sieving. The extracted protein bound neither to laurylmaltoside nor to C12E8 micelles. Its sedimentation coefficient (D20,w = 9.6 x 10(-11) m2 x s-1), diffusion coefficient (s20,w = 2S), an deduced molecular mass (20.0 +/- 1.7 kDa) are those expected for the monomeric protein. We conclude that the Rieske protein is extrinsic and therefore does not cross the membrane, although its association with the rest of the complex involves primarily hydrophobic interactions, and that the other four subunits analyzed are intrinsic.
Breyton
C
C
Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique URA 1187, Paris, France.
de Vitry
C
C
Popot
J L
JL
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D003573:Cytochrome b Group / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D003902:Detergents; D004058:Diffusion; D014450:Electron Transport Complex III; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D007506:Iron-Sulfur Proteins / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008823:Micelles; D015800:Protozoan Proteins / Q000378:metabolism
D003573:Cytochrome b Group; D003902:Detergents; D007106:Immune Sera; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D008823:Micelles; D015800:Protozoan Proteins; C037989:Rieske iron-sulfur protein; C075416:petG protein, Chlamydomonas reinhardtii; D045346:Cytochrome b6f Complex; D014450:Electron Transport Complex III