Probing for the interaction of the 32 kDa-Q B protein with its environment by use of bifunctional cross-linking reagents

Abstract

Chlamydomonas reinhardtii thylakoid polypeptides were cross-linked with hydrophobic or hydrophilic homoor hetero-bifunctional reagents, including cleavable cross-linkers containing a dithiol group (3,3-dithiobis(succinimidylpropionate), N-succinimidyl-3-(2-pyridyldithio)propionate and 3,3-dithiobis(sulfosuccinimidylpropionate), abbreviated to DTSP, SPDP and DTSSP, respectively) or glutaraldehyde. None of these reagents cross-linked the 32 kDa-Q B-protein, as identified by specific radioactive labeling and reaction with antibodies. Treatment of thylakoids with β- d-octylglucoside prior to addition of all the cross-linkers used, resulted in effective cross-linking of the 32 kDa-Q B-protein. Treatment of thylakoids with cross-linkers, followed by removal of the non-reacted reagents and addition of β- d-octylglucoside, caused extensive cross-linking of the 32 kDa-Q B-protein by SPDP and, to a lesser extent, by DTSP, indicating a possible formation of monovalent links with the 32 kDa-Q B-protein in the native membrane. The 32 kDa-Q B-protein was not cleaved by trypsin in thylakoids treated with DTSP, DTSSP or glutaraldehyde, while SPDP did not have a similar protective effect. Photosystem II activity and the affinity for Diuron binding and/or the number of binding sites were also affected by cross-linkers. None of the cross-linkers prevented the light-induced degradation of the 32 kDa-Q B-protein in isolated thylakoids exposed to high light intensity. It is suggested that the 32 kDa-Q B-protein is confined within a specific hydrophobic environment which might be essential for its function and prevent the cross-linking of the 32 kDa-Q B-proteins −SH or −HN 2 groups with similar reactive groups of neighboring proteins. Alteration of the membrane lipid phase by detergent treatment allows cross-linking to occur.