Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein.

C Breyton,C De Vitry,J.L Popot

doi:10.1016/s0021-9258(17)37329-5

C Breyton, C De Vitry + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)37329-5

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Abstract

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.

Highlights

The mode of membrane attachment of five subunits their spanning the membrane with 4,1,3, and 1transmemfrom Chlamydomonas reinhardtii cytochrome be f com- branea-helices,respectively(seeSzczepaniak et al(1991), plex has been studied using biochemical approaches
We conclude that the Rieske protein is extrinsic and of biochemical approaches.We conclude that the Rieske protein does not cross the membrane, its association with the rest of the complex involves primarily hydrophobic interactions, and that the other four subunits analyzed are intrinsic
An Antiserum against the NH2 Terminus of the Chloroplast Rieske Protein-An antiserum raised against a synthetic peptide with the sequence of the N H 2 terminus of the mature Rieske protein from C. reinhardtii' cross-reacts with the chloroplast Rieske protein in immunoblots of thylakoid membranes from C. reinhardtii wild-type strain, and does not label membranes from a mutant lacking the b6 f complex (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Chrome) inthe photosynthetic membranesof plants, algae, and Materzals-Sources of chemicals were the same as described by Atsome bacteria (see Malkin (1992) and Hope (1993A)).homolo- teia et al (1992),except that urea was from Tebu, SDS from Pierce, gous complex, cytochrombe el, plays a similar role in mitochon- ClzEs and laurylmaltoside fromCalbiochem. Determination of Sedimentation Coefficients-The supernatant of a 2 M KSCN extraction, or the Rieske protein dissociated from partially purified b6fcomplex by delipidation on a hydroxylapatite column,' supplemented or not witheither 0.02% C12Esor 0.03% laurylmaltoside (LM),were dialyzed overnight a t 4 "C against 1liter of 20 m~ Tricind NaOH buffer, pH 8.0, containing protease inhibitorsand thesame concentration of detergent. Thylakoid membranes from wild-type C. reinhardtii cells (wt, or from a b6f-less mutant (FuD4) were analyzed bySDS-PAGE.A, proteins stainedwith Coomassie Brilliant Blue (C.B.B.).B, immunoblot labeled with an antiserum raised against the NH2 terminus of the mature chloroplast Rieske FeS protein and revealed using lZ51-protein A. densities of the two detergents in sucrose gradients have been determined previously to be 1.022 and 1.167, respectively (de Vitry et al, 1991).Sedimentation coefficientwas calculated not to be affectedby the binding of up to 1micelle of CI2Es.Binding of either 20 or 98 molecules of LM was calculated to increases to 2.3 and 3.4 S, respectively.

RESULTS

DISCUSSION

11.3. Speciessequence differences