Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides

Abstract

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the a and ,B polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the a polypeptide and reduced the A875 by 50%. Proteinase K cleaved the , polypeptide of chromatophores and the a polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the 18 polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the a polypeptide was intact. We concluded that the N terminus of the 18 polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the a polypeptide is exposed on the periplasmic surface.The photosynthetic apparatus of Rhodobacter sphaeroides is found in an intracellular membrane system formed by invagination of the cytoplasmic membrane (6). Isolated membrane fractions contain several major polypeptides involved in the harvesting and use of light energy. The most abundant of these fractions are the bacteriochlorophyll aassociated polypeptides of the B800-850 and B875 lightharvesting complexes, which are named according to the wavelengths of light which they absorb (5, 6). These complexes harvest incident light and pass energy downhill to photochemical reaction centers. There, the energy is trapped and converted to an electrochemical potential across the membrane (6).The R. sphaeroides B875 complex contains two polypeptides, a and ,B, with 1:1 stoichiometry and molecular weights of 6,800 and 5,500, respectively (2, 18). Two moles each of bacteriochlorophyll a and carotenoid pigments are associated with these per each a and ,B polypeptide (2, 5). B875 complexes (and B870 complexes in related organisms) serve as energy transfer intermediaries between B800-850 complexes, carotenoids, and reaction centers (4,10,17). This role depends on the location and orientation of B875 in the lipid bilayer and its physicdl relationship to the other pigment-protein complexes.Comparatively little is known about the membrane topography of B875 in R. sphaeroides. Spectroscopic and membrane fusion studies indicate that B875 and B800-850 are laterally arranged in the membrane so that they are connected through the energy pathway (10, 17). Recently, the amino acid sequences of the a and a polypeptides have been determined, and this information has been used to speculate on the transverse arrangement of these polypeptides in the membrane (18). For example, in both polypeptides, a central domain containing a putative bacteriochlorophyll a binding site (histidine residue) and consisting of 20 hydrophobic amino acids is flanked by N-and C-terminal hydrop...

Examination of the putative Ca2+-binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium Thermochromatium tepidum

“…-ATPase. It is well established that the F 1 part of ATPase is localized on the outer surface of chromatophore (Oelze 1978;Takemoto and Bachmann 1979), whereas the C-terminal portions of all the LH polypeptides are orientated to the internal space of the chromatophores Takemoto et al 1987;Tadros et al 1988). Under the condition of this study, EDTA was essentially unable to penetrate into the membrane vesicles, but the membrane can be easily solubilized by the detergents.…”

“…It has been well established that the chromatophore has an inside-out orientation where the cytoplasmic inner surface of intracytoplasmic membrane is exposed to the external medium (Oelze and Drews 1972;Matsuura and Nishimura 1977;Lommen and Takemoto 1978;Oelze 1978;Takemoto and Bachmann 1979). The methods used for identifying the orientation of chromatophore have also been applied to determine the membrane topology of LH and RC polypeptides Takemoto et al 1987;Tadros et al 1988;Pugh et al 1998). It is shown that the N-termini of all LH polypeptides, including PufX, are exposed to the cytoplasmic surface of membrane, while the C-termini are exposed to the periplasmic membrane surfaces.…”

Consequences for the Organization of Reaction Center-Light Harvesting Antenna 1 (LH1) Core Complexes of Rhodobacter sphaeroides Arising from Deletion of Amino Acid Residues from the C Terminus of the LH1 α Polypeptide

“…Although at present little is known about the assembly pathway of the LH1 complex, topology studies have demonstrated that the C-terminal regions of both the ␣ and the ␤ polypeptides are located on the periplasmic side of the membrane (25). Thus it is conceivable that truncations of the C terminus of the ␣ polypeptide would affect the process by which it is translocated across the bilayer, leading to a decrease in the level of the ␣ polypeptide in the membrane, accompanied by a decrease in the stability of the ␤ polypeptide.…”

ISOLATION AND CHARACTERIZATION OF A STRUCTURAL SUBUNIT FROM THE CORE LIGHT‐HARVESTING COMPLEX OF Rhodobacter sphaeroides 2.4.1 AND puc705‐BA

“…The polypeptides of the core LHC of these three bacteria have been sequenced and are similar in hydrophobicity and molecular weight (Brunisholz and Zuber, 1988;Zuber, 1986). These polypeptides are membrane-spanning with their N-termini near the cytoplasmic surface and their C-termini near the periplasmic surface (Brunisholz et al, 1984;Tadros et al, 1986a, b;Takemoto et al, 1987). Reconstitution results strongly suggest that BChl binding in "B820" complexes is primarily to the p polypeptides and Rs.…”