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 sphaero ides in which the pufX gene had been deleted, it was possible to ident ify by HPLC membrane protein components present in pufX(+) cells but a bsent in pufX(-) cells. In parallel preparations, membrane proteins so luble in chloroform/methanol containing ammonium acetate were first ex tracted from lyophilized membrane fractions of the pufX(+) cells and s eparated from pigments and larger protein material by gel-filtration c hromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX(+) material that were not pres ent in pufX(-) material. From N-terminal amino acid sequencing, the Pu fX protein of Rb. capsulatus was identified, and from positive interac tion with a PufX protein antibody, the Rb. sphaeroides PufX protein wa s identified. Although overall yields were very small, sufficient quan tities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subu nit complex. From the behavior of the PufX protein and the ol-polypept ide of LH1 on HPLC, qualitative evidence was obtained that the two pro teins have a high affinity for each other. In reconstitution assays wi th bacteriochlorophyll (Bch1) 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 exhibi ted by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bch 1 and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the cl-pol ypeptide causing 50% inhibition were approximately 0.5. Formation of t he heterologous (alpha beta) subunit-type complex formed with Bch1 and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also in hibited by low concentrations of the Rb. capsulatus PufX protein (appr oximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). Howe ver, neither PufX protein inhibited formation of a homologous (beta be ta) subunit-type complex, which indicates that the PufX proteins do no t interact with the beta-polypeptides.