The Light-Harvesting Polypeptides ofRhodospirillum rubrum.II. Localisation of the Amino-Terminal Regions of the Light-Harvesting Polypeptides B 870-α and B 870-β and the Reaction-Centre Subunit L at the Cytoplasmic Side of the Photosynthetic Membrane ofRhodospirillum rubrumG-9+

Abstract

The nonspecific proteinase K and the specific proteases .alpha.-chymotrypsin, trypsin and Staphylococcus aureus V8 protease were used to determine the orientation of the polypeptides B870-.alpha. and B870-.beta. from the major antenna complex B870 of R. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B870-.alpha. exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting in B870-.beta. only within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B870-.alpha.) and 16 (B870-.beta.) amino acid residues were removed from their N-terminal parts as proved by Edman degradation of cleavage products. The major peptide bonds cleaved were identified as .**GRAPHIC**. B870-.alpha. and as .**GRAPHIC**. in B870-.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 B870-.alpha. and B870-.beta. is postulated, with their N-termini 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., in which the hydrophobic stretch of B870-.alpha. and of B870-.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 MW of 14,000 daltons. Its N-terminal amino acid sequence is identical with the sequence within the N-terminal region of the reaction center subunit L of R. rubrum G-9+. Thus, as in the case of B870-.beta., proteinase K probably removed 16 amino acid residues from the N-terminal part of subunit L. Apparently, this subunit is also exposed at the surface of the cytoplasmic side of the chromatophore membrane.