Orientation of the B800-850, B870, and reaction center polypeptides on the cytoplasmic and periplasmic surfaces of Rhodobacter capsulata membranes

Tadros, Monier H.; Frank, Rainer; Doerge, Bernhard; Gad’on, Nasser; Takemoto, Jon Y.; Drews, Gerhart

doi:10.1021/bi00398a022

Cited by 37 publications

(39 citation statements)

References 36 publications

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“…The LHI apoproteins are integral proteins of the intracytoplasmic membrane (ICM) and span the membrane once with * Corresponding author. a central hydrophobic domain (51)(52)(53). The NH2 terminus of the LHI a polypeptide is located in the cytoplasm and has a short hydrophobic central part of amino acid residues flanked by positively charged residues which are next to a conserved proline (51,52).…”

Section: )mentioning

confidence: 99%

“…a central hydrophobic domain (51)(52)(53). The NH2 terminus of the LHI a polypeptide is located in the cytoplasm and has a short hydrophobic central part of amino acid residues flanked by positively charged residues which are next to a conserved proline (51,52). The LHI a protein is inserted into the ICM in wild-type amounts only in the presence of the LHI 1 protein (43).…”

Section: )mentioning

confidence: 99%

“…Mutant strains of R. capsulatus were grown in malate mineral medium (18) Analytical methods. ICMs of R. capsulatus strains were purified by discontinuous sucrose gradient centrifugation as described elsewhere (28,51). Total amounts of the pigments Bchl and carotenoids were determined according to Clayton (10).…”

Section: )mentioning

confidence: 99%

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Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants

1992

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The NH2 termini of light-harvesting complex I (LHI) polypeptides a and ,B of Rhodobacter caus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI a protein in insertion into the intracytoplasmic membrane (ICM) of R. ca,psulaus, amino acids 6 to 8, 9 to 11, 12 and 13, or The LHI a and 1 polypeptides are present in a 1:1 ratio. The LHI apoproteins are integral proteins of the intracytoplasmic membrane (ICM) and span the membrane once with * Corresponding author. a central hydrophobic domain (51-53). The NH2 terminus of the LHI a polypeptide is located in the cytoplasm and has a short hydrophobic central part of amino acid residues flanked by positively charged residues which are next to a conserved proline (51, 52). The LHI a protein is inserted into the ICM in wild-type amounts only in the presence of the LHI 1 protein (43). The LHI 1 protein, however, has a negatively charged NH2 terminus and is able to integrate temporarily into the membrane in the absence of the LHI a protein (43). The LHI 13 protein was observed to be inserted earlier into the ICM than was the LHI a protein (43).The described structures and features of the LHI a and 1 polypeptides are conserved among several species of the genus Rhodospirillaceae (50). Two highly conserved amino acid residues (Trp-8 and Pro-13) are present in the NH2 terminus of the LHI a polypeptide (50). The aromatic structure of the Trp at position 8 seems to be of importance for the insertion of the LHI a polypeptide into the ICM (42). The conservative amino acid residue Pro-13, however, seems to be necessary for keeping the LHI 1 partner polypeptide in the membrane (42).Deletions or insertions have been introduced into the NH2 terminus of the LHI a protein to obtain more information on the structural requirements for the membrane insertion of the LHI a protein and the assembly of both LHI proteins into a functional complex. The construction and characterization of R capsulatus LHI-strains containing a mutated LHI a polypeptide are described in this report. Additionally, LHI+ revertant strains of one LHI-mutant strain are characterized.3030

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confidence: 99%

Section: )mentioning

confidence: 99%

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Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants

1992

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“…To understand the topography and organization of the pigment-protein complexes and the insertion and assembly of polypeptides in the membrane, the orientations of the N-and C-terminal domains and conserved residues in these regions are of importance. On the basis of protease degradation experiments and amino acid sequence analysis, a transmembrane orientation with the N termini facing the cytoplasm was postulated for B870a, B870p, and RC-L of Rhodospirillum rubrum (8,9) and for B800-850cx, B800-850p, B870a, B870p, and RC-L of Rhodopseudomonas capsulata (27)(28)(29) and R. sphaeroides (32). In this study, the orientations of the N and C termini of RC-L, RC-M, and LH polypeptides of R. sphaeroides were studied.…”

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Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides

et al. 1988

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The localization of the N-and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850a and B870a polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850a and B870a polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the a polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850,1 polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the , polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the B polypeptides are believed to point to the periplasmic space.The photosynthetic apparatus of Rhodobacter sphaeroides is located in an intracytoplasmic membrane system formed by invaginations of the cytoplasmic membrane (15). The major components of the photosynthetic apparatus are the light-harvesting (LH) or antenna pigment-protein complexes and the photochemical reaction center (RC), which are present as integral membrane particles (14). The absorption of light energy by LH complexes creates excited singlet states which migrate over the antennae downhill to the RC where they are trapped and converted into a membrane potential difference and a redox potential difference. This conversion drives a cyclic electron transport and the formation of a proton gradient across the membrane (16).The bacteriochlorophyll and carotenoid pigments are bound in stoichiometric ratios to the polypeptides of the three complexes: RC and the LH complexes B870 and B800-850, named according to their near-infrared absorption bands. In each LH complex, two different polypeptides bind noncovalently two or three bacteriochlorophyll and one or two carotenoid molecules. The axes of the pigment molecules have a strict orientation relative to the plane of the membrane (6, 18, 25). The pigment-binding polypeptides span the membrane once by a transmembrane a helix of hydrophobic amino acyl residues in LH complexes (5). The RC subunits M and L (RC-M and RC-L, respectively) span the membrane five times each (1,13,22,39). The N-and C-terminal domains of L...

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“…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 cyto- plasm (34). None of the eight polypeptides is synthesized with a cleavable, NH2-terminal signal sequence (8).…”

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

Troschel

Müller

1990

The Journal of Cell Biology

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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 (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 B800-850 (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 cyto-1. 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 ...

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Orientation of the B800-850, B870, and reaction center polypeptides on the cytoplasmic and periplasmic surfaces of Rhodobacter capsulata membranes

Cited by 37 publications

References 36 publications

Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants

Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides

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

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