Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex.

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.