Study of the Structure of the Thylakoid Membrane-Bound Chloroplast Coupling Factor CF₁

A. N. Malyan^1*, O. I. Vitseva¹, and H. Strotmann²

¹Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-0967) 79-0532; E-mail: Malyan@issp.serpukhov.su

²Institute of Plant Biochemistry, Duesseldorf University, Germany

^* To whom correspondence should be addressed.

Received December 2, 1998; Revision received April 1, 1999

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The structure of thylakoid membrane-bound chloroplast coupling factor CF₁ was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21--E22. The cleavage of this peptide bond caused the alphaK24--V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14--E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140--S141, alphaG160--R161, and betaG102--G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF₁ is more sensitive to proteolysis than that of solubilized CF₁. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17--G18 and alphaE22--V23 bonds in isolated CF₁. These results suggest a significant structural difference between soluble and membrane-bound CF₁. A number of peptide bonds, alphaG160--R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160--R161 bond.

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KEY WORDS: chloroplasts, coupling factor CF₁, ATPase, ATP synthase, protein structure, proteolysis

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