Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus.

Abstract

A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described. Synthesis of two proteins of the bacterium's photosynthetic apparatus (light-harvesting complex B870 alpha and beta) was performed by SP6 polymerase transcription of the subcloned genes, isolation of the mRNA and translation in vitro using a cell-free extract of R. capsulatus cells. The integration of these proteins in vitro into added intracytoplasmic membrane vesicles (ICM) is demonstrated. Without addition of ICM approximately 70% of the synthesized B870 proteins were soluble. If, however, ICM were present during synthesis, the majority of the soluble protein was found to associate with the membranes. The membrane-associated polypeptides could be solubilized only by detergent treatment but could not be extracted by treatment at alkaline pH (Na2CO3), suggesting that the proteins had been firmly inserted into the lipid bilayer. Moreover, the B870 alpha and beta proteins that integrated in vitro into ICM were also found to associate with pigment ligands and to assemble into a native reaction center/B870 complex. The native conformation of this complex isolated from ICM by Triton fractionation was demonstrated by microspectral analysis of the bound pigments.