Localization and orientation of subunit delta of spinach chloroplast ATP-synthase within the CF0 CF1 complex. 2. Identification of C-terminal residues of delta, exposed on the thylakoid membrane.
The amino acid residues of spinach CF1 subunit delta are identified which are accessible and thus exposed within the quaternary structure of the ATP-synthase complex on the thylakoid membrane. Two types of antibodies in the monospecific polyclonal antiserum 306 against CF1 delta, described in the previous publication [Z. Naturforsch. 44c, 153-160 (1989)], were separated by virtue of their different affinity to thylakoid membranes and used for specific analysis of the products of proteolytic digestion of delta in situ. Polypeptide delta in situ, i.e. within the CF0 CF1 complex on the membrane, is not susceptible to digestion by aminopeptidase M and trypsin, but is shortened by about 1 kDa by carboxypeptidase Y and digested at residues Glu173 and Glu179 by the Staphylococcus aureus protease V8. The epitope on delta reacting with the agglutinating antibodies from serum 306 is lost after these proteolytical treatments and therefore situated on residues Met180-Val187. Since trypsin destroys this epitope only after prolonged incubation and with at least 50 micrograms trypsin/mg Chl, residue Lys169 of delta probably is inaccessible in situ. We conclude that the C-terminal amphipathic alpha-helix of spinach CF1 subunit delta is exposed on the thylakoid membrane, with the hydrophilic face directed to the outside, and that CF1 delta starts to be shielded within the quaternary structure of the CF0 CF1 complex between Glu173 and Lys169. The hydrophobic face of the c-terminal helix may be part of the binding surface towards CF0. Antibodies from serum 306 inhibit the PMS mediated cyclic photophosphorylation by reacting with C-terminal residues of delta.
Berzborn
R J
RJ
Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie, Ruhr-Universität Bochum, Bundesrepublik Deutschland.
Finke
W
W
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D000939:Epitopes / Q000032:analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D000939:Epitopes; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases