Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype

Lilburn, Timothy; Prince, Roger C.; Beatty, J. Thomas

doi:10.1128/jb.177.16.4593-4600.1995

Cited by 36 publications

(27 citation statements)

References 32 publications

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“…Furthermore, the third phase of the bandshift (compare the two panels of Fig. 5b) appeared to be reduced in MA05, suggesting that the reduced amount of RC in MA05 is impaired in quinol transfer from the RC to the cytochrome b/c 1 complex, which is characteristic of a PufX deficiency (28,29).…”

Section: The Entire Population Of Puhbmentioning

confidence: 98%

“…The puf operon is part of a superoperon that includes pigment biosynthesis genes (53) and also encodes PufQ, a regulatory factor in BChl biosynthesis (4). PufX has been implicated in the exchange of quinone/quinol between the RC and cytochrome b/c1 complexes (28,29).In this study, we investigate the basis of the RC/LH1 deficiency and impaired photosynthetic growth of R. capsulatus puhB disruption strains by examining the effects of PuhB on RC assembly in the absence of LH1, LH1 assembly in the absence of the RC, and the abundance of PufX. We also evaluate the ability of each of the three putative transmembrane (TM) segments of PuhB to span the inner membrane and to self-associate.…”

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confidence: 99%

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The PuhB Protein of Rhodobacter capsulatus Functions in Photosynthetic Reaction Center Assembly with a Secondary Effect on Light-Harvesting Complex 1

2005

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The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.Purple nonsulfur photosynthetic bacteria such as Rhodobacter capsulatus are capable of aerobic respiratory and anaerobic photosynthetic growth. The photosynthetic apparatus includes three membrane-bound pigment-protein complexes: the reaction center (RC), which functions as a light-dependent quinone reductase; light-harvesting complex 1 (LH1), which is adjacent to and forms a ring or arc encircling the RC as part of the so-called core complex that includes the PufX polypeptide (17,22,36,38,43,51); and LH2, which is thought to be present in multiple copies of a ring-shaped structure that interconnect core complexes (33). These complexes are located within differentiated invaginations of the cytoplasmic membrane, called the intracytoplasmic membrane system, formed in response to oxygen deprivation (14). The presence of the various photosynthetic complexes can be evaluated by their characteristic light absorption spectra, which reflect the protein environments around bacteriochlorophyll a (BChl); unbound BChl has a far-red absorption peak at 780 nm, whereas this peak of the LH2 BChls of R. capsulatus is at 800 and 850 nm, and BChls in the LH1 complex absorb light at approximately 870 nm. The far-red absorption peaks of the RC are at about 760 nm (bacteriopheophytins), 804 nm (accessory or "voyeur" BChls), and 865 nm (the "special pair" of BChls) (16).In R. capsulatus, one of the three polypeptides of the RC, RC H, is encoded by the puhA gene that is transcribed as part of the bchFNBHLM-lhaA-puhABC superoperon from two promoters, one 5Ј of bchF and the other within the lhaA gene (3,6,57). A segment of the puh operon is shown in Fig. 1a. The PuhB protein (formerly known as Orf214) is required for optimal RC/LH1 l...

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Section: The Entire Population Of Puhbmentioning

confidence: 98%

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The PuhB Protein of Rhodobacter capsulatus Functions in Photosynthetic Reaction Center Assembly with a Secondary Effect on Light-Harvesting Complex 1

2005

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“…Crystal structures of the reaction center and light-harvesting (LH) integral membrane pigment-protein complexes from several species have been published (15,17,22). Although the structural proteins of these complexes are very hydrophobic and might be thought of as independently capable of complex assembly in the cytoplasmic membrane, several genes that do not encode crystal structural components and yet are required to obtain wild-type levels of the reaction center and LH complexes have been discovered (2,12,19,31). The results of preliminary experiments with one of these genes, F1696, indicate that it functions to enhance assembly of LH complex I (LHI) as opposed to inhibiting turnover (31a).…”

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confidence: 99%

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II

Leblanc

Beatty

1996

J Bacteriol

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A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC::phoA gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yielded approximately 1/20th as much activity. Replacement of phoA with lacZ in nine of the fusions confirmed the topology, as ␤-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membranespanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus ⌬pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.Such purple nonsulfur photosynthetic bacteria as Rhodobacter capsulatus are good model systems for investigations of membrane protein structure and function, especially in regard to proteins involved in photosynthesis. Crystal structures of the reaction center and light-harvesting (LH) integral membrane pigment-protein complexes from several species have been published (15,17,22). Although the structural proteins of these complexes are very hydrophobic and might be thought of as independently capable of complex assembly in the cytoplasmic membrane, several genes that do not encode crystal structural components and yet are required to obtain wild-type levels of the reaction center and LH complexes have been discovered (2,12,19,31). The results of preliminary experiments with one of these genes, F1696, indicate that it functions to enhance assembly of LH complex I (LHI) as opposed to inhibiting turnover (31a). The current evidence indicates that the protein components of pigment-protein complexes are capable of membrane association in the absence of bacteriochlorophyll, but complexes are not formed, and the proteins exhibit enhanced turnover rates (10).The crystal structure of the Rhodopseudomonas acidophila LHII complex was found to consist of a ring containing nine copies of a membrane-spanning ␣-␤ protein dimer, with bacteriochlorophyll and carotenoid pigments sandwiched between concentric transmembrane domains of the ␣ and ␤ proteins (22). The R. capsulatus LHII complex was purified and found to contain a third protein, designated ␥, which was removed from the complex by treatment with Na 2 CO 3 , with little effect on LHII light absorption properties (27). The LHII proteins are coded for by the pucBACDE operon, which has been cloned, sequenced, and studied by deletion-complementation analyses...

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“…sphaeroides . The finding was further supported by the localization of second-site suppressor mutants in the pufBA genes encoding the LH 1-α-and β-apoproteins (Lilburn et al 1995), and the lack of a PufX requirement in the absence of LH 1 as shown in Neil Hunter's laboratory (McGlynn et al 1994). These findings have suggested that in the absence of PufX, LH 1 tightly surrounds the reaction center and impedes UQ/UQH 2 movements and that the suppression of this defect results from a destabilization of LH 1, enhancing UQ exchange.…”

Section: The Assembly Of Supercomplexesmentioning

confidence: 73%

Membrane biogenesis in anoxygenic photosynthetic prokaryotes

Drews

Niederman

Discoveries in Photosynthesis

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Following the discovery of photosynthetic bacteria in the nineteenth century, technical developments of the 1950s led to their use in membrane biogenesis studies. These investigations had their origins in the isolation of subcellular particles designated as 'chromatophores' by Roger Stanier and colleagues, which were shown to be photosynthetically competent by Albert Frenkel, and to originate from the intracytoplasmic membrane (ICM) continuum observed in electron micrographs. These ultrastrucutral studies by the G. Drews group, Germaine Cohen-Bazire and others also suggested that the ICM originates by invagination of the cytoplasmic membrane, as later established in the biochemical and biophysical work of the R. Niederman and Drews groups. Through a combination of genetic approaches, first introduced in the early 1980s by Barry Marrs, and the atomic resolution structures determined for light-harvesting antennae and reaction centers, a detailed understanding is emerging of mechanisms regulating their levels in the membrane and the roles played by specific protein domains and additional factors in their assembly and supramolecular organization. Prospects for additional progress during the twenty-first century include further elucidation of molecular aspects of the assembly process and the application of newer spectroscopic probes to photosynthetic unit formation.

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Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype

Cited by 36 publications

References 32 publications

The PuhB Protein of Rhodobacter capsulatus Functions in Photosynthetic Reaction Center Assembly with a Secondary Effect on Light-Harvesting Complex 1

The PuhB Protein of Rhodobacter capsulatus Functions in Photosynthetic Reaction Center Assembly with a Secondary Effect on Light-Harvesting Complex 1

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II

Membrane biogenesis in anoxygenic photosynthetic prokaryotes

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