The light-harvesting polypeptides of Rhodospirillum rubrum. II. Localisation of the amino-terminal regions of the light-harvesting polypeptides B 870-alpha and B 870-beta and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+.
The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.
D000596:Amino Acids / Q000032:analysis*; D020130:Bacterial Chromatophores / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification; D003593:Cytoplasm / Q000032:analysis*; D019286:Endopeptidase K; D010450:Endopeptidases; D006868:Hydrolysis; D010447:Peptide Hydrolases; D010455:Peptides / Q000302:isolation & purification; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins; D012247:Rhodospirillum rubrum / Q000032:analysis* / Q000254:growth & development / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D001426:Bacterial Proteins; D010455:Peptides; D045322:Photosynthetic Reaction Center Complex Proteins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D019286:Endopeptidase K