Topography of the Chloroplast Cytochrome b6: Orientation of the Cytochrome and Accessibility of the Lumen-Side Interhelix Loops
A. Szczepaniak, M. T. Black, W. A. Cramer
Zeitschrift für Naturforschung C - A Journal of Biosciences
The to pography of chloroplast cytochromes f and b6 was probed with proteases carboxypeptidase A (CpA ), trypsin, and Staph, aureus V8. The cytochrome and its proteolytic products were detected by heme stain and, in most experiments, by immunoreaction. In thylakoids, the only protease that significantly affected the intactness of cytochrome f was CpA that caused a small (ΔMr = -1 - 2000) decrease in the apparent molecular weight. In SDS -treated thylakoids, both trypsin and V8 degraded
... e f. The inferred topography of cytochrom f , with the COOH -terminus on the stromal (n) side, one membrane-spanning a-helix n ear the COOH-terminus, and most of the Cyt f mass on the lumen (p) side, is consistent with that previously inferred by others. Cytochrome b6 was not sensitive to CpA . but was more sensitive to trypsin and V8 protease than cytochrome f , cytochrome b-559, or the 17 kDa OEC extrinsic protein. Trypsin caused a small decrease in size of cytochrome b6, which was observed using whole protein antibody as a single smaller band (ΔMr ≈ 2000) or two smaller discrete bands (ΔMr = -1000 and 2500, respectively) which, unlike the untreated protein , did not react with antibody generated to a peptide mimicking A sp-5 -Gln-14 near the NH2-terminus. These shorten ed tryptic fragments were attributed to cleavage after R-10 and K-23 n ear the NH2-terminus, implying an orientation with the NH2-terminus on the stromal side of the membrane. The sensitivity of cytochrome b6 toward this trypsin cleavage was increased if the membranes were first incubated with CpA , showing that the NH2 -terminal region of cytochrome b6 is masked by the COOH -terminal domain of one or more thylakoid proteins. Under conditions where degradation of cytochrome b-559, cytochrome f, or the 17 kDa OEC extrinsic protein on the lumen side of the membrane was small ( < 10-20% ). V8 protease degraded 80-90% of cytochrome b6. The main polypeptide fragment generated by V8 protease had an apparent molecular weight Mr = 16,000, reacted with antibody to a peptide mimicking Ala-146-Asp-155, and was more pronounced in membranes treated with SDS. The fragment could then result from cleavage at two sites, Glu-74 and /or Glu-166. It is concluded that the loop of cytochrome b6 protruding from the lumen side of the membrane between helices III and IV , and possibly that between helices I and II as w ell, contains a single Glu residue conferring a sensitivity to V8 protease greater than that of the lumen-side mass of cytochrome f which has many more Glu residues. This implies a lumen-skeletal structure in which one or two loops of cytochrome b6 on the lumen side are more exposed to the aqueous phase than cytochrome f.