Development of a Cell-free System to Study the Membrane Assembly of Photosynthetic Proteins of Rhodobacter capsulatus

A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described. Synthesis of two proteins of the bacterium's photosynthetic apparatus (lightharvesting complex B870 ot and/~) 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 ,,070% 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 t~ and 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. T HF. Gram-negative, facultatively photoheterotrophic bacterium Rhodobacter capsulatus is a good model organism for studying membrane differentiation because of its inducible apparatus for photosynthesis. The assembly of the photosynthetically active intracytoplasmic membrane (ICM) ~ system is induced by lowering either the oxygen tension or the light intensity (reviewed in references 9, 17, 40). The photosynthetic complexes are composed of eight different integral membrane proteins, organized into two light-harvesting (LH) complexes, B870 (LH-I) and B800850 (LH-II), and a reaction center (RC). Each LH-complex consists of two pigment-binding proteins in a 1:1 stoichiometry; B870 t~ and/3 (Mr = 12 and 7 kD), and B800-850 ot and ~ (Mr = 10 and 8 kD). The B800-850 complex also contains the non-pigment-binding protein V (Mr = 14 kD). Each of these ot and/3 peptides has one et-helical transmembrane segment of • 20 amino acids with the NH2 terminus facing the cytoplasm and the COOH terminus located in the periplasmic space (32-34). The RC consists of the two pigment-binding proteins L and M (Mr = 20.5 and 24 kD), each possessing five membrane-spanning segments, and the non-pigment-binding protein H (Mr = 28 kD), which is anchored within the membrane by a single hydrophobic stretch. The three RC proteins are found in the ICM in a 1:1:1 stoichiometry with their NH2 termini located in the cyto1. Abbreviations used in this paper: ICM, intracytoplasmic membrane vesicles; Me2SO, dimethyl sulfoxide; TeaOAc, triethanolamine acetate. plasm (34). None of the eight polypeptides is synthesized with a cleavable, NH2-terminal signal sequence (8). The genes for the eight proteins of the photosynthetic complexes are organized into three operons: puf(B870 a and ~, RC-L and M), puh (RC-H), and puc (B800-850). All of the structural genes have been sequenced (35, 38, 39). Little is known about the assembly mechanism of the photosynthetic complexes. It was shown that (a) they appear to be inserted into distinct membrane areas (7, 15, 29); (b) the RC is not properly assembled if the B870 complex is missing (14); (c) pigments are required for the stability and proper assembly of the protein complexes (20). The use of cell-free systems greatly facilitates the investigation of the molecular mechanism of protein integration into membranes. These systems allow the selective expression of individual proteins whose membrane transport can be reconstituted by the addition of exogenous membrane vesicles. Once such a system is established, the transport mechanism can be studied in more detail with the eventual aim of identifying molecular components of this process. Up to now, in prokaryotes, such an analysis using cell-free translation-transport systems has only been carried out for the plasma membrane of Escherichia coli (for a recent review see reference 30). Here we report the development and characterization of a cell-free system from R. capsulatus for the study of the membrane integration of newly synthesized pigment-binding proteins. The two B870 proteins were synthesized in vitro by programming a membrane-free extract from R. capsulatus with © The Rockefeller University Press, 0021-9525/90/07/87/8 $2.00 The Journal of Cell Biology, Volume 111, July 1990 87-94 87 on May 2, 2019 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.111. 1.87 Published Online: 1 July, 1990 | Supp Info: