In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides

Abstract

The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo-with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.When Rhodopseudomonas sphaeroides is grown chemoheterotrophically in the presence of oxygen, it is a typical gram-negative bacterial cell; when the oxygen tension is reduced below threshold levels, the cell responds by synthesizing an extensive intracytoplasmic membrane system (13). This new membrane houses the numerous components of the photosynthetic apparatus. In addition to regulation by oxygen tension, the synthesis of the components necessary for photophosphorylation is also modulated by light intensity (19). Given these attributes, the photosynthetic bacteria, and R. sphaeroides in particular, have been employed as a model system for the study of membrane biosynthesis and photosynthesis at the molecular level (20).One of the key complexes within the intracytoplasmic membrane is the photosynthetic reaction center (RC). The RC used light energy in the formation of high-energy electrons, and it is these electrons which are the driving force of photosynthetic electron transport. Studies have compared R. sphaeroides RC with the photosystem II RC of chloroplasts (17). Three integral membrane protein subunits, designated H, M, and L, make up the R. sphaeroides RC. In addition to the three polypeptides, there are four bacteriochlorophylls, two bacteriopheophytins, two quinones, and one iron in a functional RC (27). Polypeptides H, M, and L are extremely hydrophobic and have apparent Mrs of 28,000, 24,000, and 21,000, respectively, based on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, although these are not the true molecular weights (27). The actual role of each subunit is unclear, but dissociation of H from L and M leaves the L-M complex with spectral and functional properties similar to those of the whole RC (10).Despite extensive biochemical analysis of the photosynthetic membranes and RCs, very little is known about the assembly of these proteins or the regul...

Cloning and expression of the Rhodobacter sphaeroides reaction center H gene

“…The in vitro-synthesized proteins from an R. sphaeroides-coupled S-30 system (5) were labeled with L-[35S]methionine (approximately 40 p.Ci per 100-,u reaction volume) and were visualized by fluorography of En3Hance-treated SDS gels with preflashed Kodak Xomat AR-5 film. SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described previously (14,15,30). Approximately 2 pug of purified plasmid DNA was used as template in these studies, and translation assays were performed for 60 min before protein synthesis was terminated with 10 pul of 250-,ug/ml chloramphenicol.…”

“…The variety of biochemical (lOa, 17,19), spectroscopic (28,29), structural (9,10), and molecular biological (14,34,35,(37)(38)(39) techniques which have been used to analyze the photosynthetic intracytoplasmic membrane (ICM) in both wild-type and mutant strains of these bacteria make the photosynthetic membrane one of the most intensively studied of all biological membranes.…”

“…The RC complex of Rhodobacter sphaeroides (recently redefined from the genus Rhodopseudomonas [16]) is an integral membrane protein complex of four molecules of Bchl, two bacteriopheophytins, an iron atom, two molecules of ubiquinone, and three polypeptide subunits, referred to as RC-H, RC-M, and RC-L, in a 1:1:1 stoichiometry (28). Numerous physical and biochemical techniques have been used to study the structure, function, and topography of the RC complex within the membrane, the binding and organization of the photopigment molecules within the RC complex, and the associations of individual RC proteins with each other (15,28,29). The recent preparation and analysis of crystals of purified RC complexes from Rhodopseudomonas virdis (9,10) and R. sphaeroides (1) show the potential for elucidating the detailed structure, organization, and functions of the various components of this complex.…”

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

“…Chory and Kaplan described a translation system based on an S-30 from R. sphaeroides (6). Only Hoger et al (13) reported membrane-association of pigment-binding proteins of Rhodobacter sphaeroides in vitro; however, their cell-free system was not clearly shown to be membrane dependent. We report here that the in vitro-synthesized proteins were found to integrate into exogenously added ICM.…”

“…Fig. 4 A, lanes 9-14 show an analogous experiment in which membrane-free protein synthesis was inhibited after 45 min by puromycin; membranes were added posttranslationally and the sample was incubated for another 30 min (lanes [12][13][14]. A control (lanes 9-1/) was treated identically except that ICM were omitted.…”