Zero-length crosslinking between subunits delta and I of the H(+)-translocating ATPase of chloroplasts.
Treatment of spinach thylakoids with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) induced formation of a zero-length crosslink of an apparent molecular mass of 38 kDa. This product was shown, by immunodetection, to consist of subunit delta of CF1 and subunit I of CF0. The crosslink was isolated by preparative SDS gel electrophoresis and subjected to cyanogen bromide cleavage. Electrophoretic and immunological analysis of the resulting peptides suggested that the crosslink was formed between a glutamyl or aspartyl residue at the C-terminal end of subunit I and a basic amino acid of subunit delta in the range between Val-1 to Met-165. Treatment of thylakoids with EDC/Sulfo-NHS resulted in inhibition of photophosphorylation and CF0CF1-catalyzed ATP hydrolysis without affecting formation of a proton gradient related to phenazine methosulfate-mediated cyclic electron transport. Inhibition of H+ transport-coupled ATP hydrolysis was more pronounced than non-coupled methanol-stimulated ATP hydrolysis. The results suggest that subunits delta and I form a connection between the partial complexes CF1 and CF0 in situ. Crosslinking of the two subunits may impede the translocation of protons through CF0CF1.
Beckers
G
G
Institut für Biochemie der Pflanzen, Heinrich Heine Universität Düsseldorf, Germany.
Berzborn
R J
RJ
Strotmann
H
H
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010944:Plants; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D006180:Proton-Translocating ATPases; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide