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

Troschel, Dorothee; Müller, Matthias

doi:10.1083/jcb.111.1.87

Cited by 25 publications

(15 citation statements)

References 40 publications

(50 reference statements)

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“…On the other hand, in vitro studies (44) demonstrated that a cotranslational membrane association was more efficient than a posttranslational one, which suggests synthesis on membranebound ribosomes.…”

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Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

Richter

Drews

1991

J Bacteriol

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The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI a and LHI 0, encoded by the pufA (LHI a) and puJB (LHI j1) genes. Pulse-labeling experiments showed that in the presence of the LHI a polypeptide, the LHI , polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI a polypeptide.Each of the pufA and puJB genes was deleted to test whether the LHI a and polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI , polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI a polypeptide was present. However, the LHI , polypeptide did not accumulate in the membrane in the absence of the LHI a protein but was degraded linearly within about 12 min. In contrast to the LHI ,1 protein, only trace amounts of the LHI a polypeptide were inserted into or attached to the membrane if the LHI , polypeptide was not synthesized.The facultative phototrophic bacterium Rhodobacter capsulatus has the ability to transduce light energy into metabolic energy. Two different light-harvesting (LH) complexes are involved in absorption of light quanta and transfer of excitation energy to the photosynthetic reaction center (RC). LH complex I (LHI) or B870 is associated with the RC forming the core complex (13, 21). LHII (B800-850) surrounds the core complex and is synthesized in variable amounts, depending on growth conditions (7, 13).The pigment-protein complexes are located in the intracytoplasmic membrane (ICM). Each complex is composed of two pigment-binding integral membrane proteins: LHI a and ,B, LHII a and 3, and RC-L and RC-M. A non-pigmentbinding protein is present in the LHII antenna (LHII -y polypeptide) and in the RC (RC-H subunit) (for reviews, see references 13 and 14). The pigments bacteriochlorophyll and carotenoids are bound noncovalently in stoichiometric ratios to the antenna and RC polypeptides (for a review, see reference 13).Pigments and pigment-binding proteins are coordinately assembled in distinct membrane areas (9) whose relative phospholipid concentration has an influence on the insertion (3,21,22,31,43 The LHI a and LHI fi polypeptides are encoded by the puf operon (1). Synthesis of these polypeptides is controlled at the mRNA level in response to changes of oxygen partial pressure or light intensity (1,5,22,50 hydrophobic a-helical central segment (39,41,51). The N-terminal segments of LHI a and LHI P polypeptides are oppositely charged (40, 42) and located on the cytoplasmic site of the membrane (41). The C termini of both LHI polypeptides extend into the periplasmic space (40). It was proposed that the LHI complex is stabilized by ionic interaction between the oppositely char...

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Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

Richter

Drews

1991

J Bacteriol

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“…the translocation of preLamB, by in-vitro-assembled SecY, provides evidence that in-vitro-synthesized SecY integrates into the lipid bilayer of INV in an enzymically active conformation. Similar assays for the functional integration of invitro-synthesized, bacterial plasma-membrane proteins have recently been described for the lactose permease of E. coli (Ahrem et al, 1989) and the subunits of light-harvesting complex I of Rhodohacter cupsulutus (Troschel and Miiller, 1990).…”

Section: Functional Membrane Integration Of Secy In Vitromentioning

confidence: 99%

“…We have recently described a method which employs sucrose-gradient centrifugation to separate free from membrane-associated integral membrane proteins following in vitro synthesis (Ahrem et al, 1989;Troschel and Miiller, 1990). Fig.…”

Section: In-vitvo-synthesized Secy Associates With Plasma-membrane Vementioning

confidence: 99%

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

Swidersky

Rienhöfer-Schweer

Werner

et al. 1992

European Journal of Biochemistry

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SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to p-lactamase and , I receptor. When SecYdeprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the , I receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the sec Y gene suppresses signal-sequence mutations of the , I receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.

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“…We have now cloned, isolated, and characterized SecA of R. capsulatus. The existence of a homologous in vitro system (58,66) to study the translocation activity of SecA enabled us to directly compare SecA of R. capsulatus with that of E. coli. It thereby became evident that the activity of a distinct SecA species depends not only on the target membranes but also exquisitely on the precursor to be translocated.…”

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“…We have started to analyze protein export in Rhodobacter capsulatus (57,58,66,67), which belongs to the group of ␣-subclass purple bacteria. Based on a 16S rRNA sequence analysis (68), these bacteria are closely related to the ancestral prokaryote that, according to the endosymbiont theory, gave rise to mitochondria.…”

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Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity

et al. 1997

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We have cloned the secA gene of the ␣-subclass purple bacterium Rhodobacter capsulatus, a close relative to the mitochondrial ancestor, and purified the protein after expression in Escherichia coli. R. capsulatus SecA contains 904 amino acids with 53% identity to E. coli and 54% identity to Caulobacter crescentus SecA. In contrast to the nearly equal partitioning of E. coli SecA between the cytosol and plasma membrane, R. capsulatus SecA is recovered predominantly from the membrane fraction. A SecA-deficient, cell-free synthesistranslocation system prepared from R. capsulatus is used to demonstrate translocation activity of the purified R. capsulatus SecA. This translocation activity is then compared to that of the E. coli counterpart by using various precursor proteins and inside-out membrane vesicles prepared from both bacteria. We find a preference of the R. capsulatus SecA for the homologous membrane vesicles whereas E. coli SecA is active with either type of membrane. Furthermore, the two SecA proteins clearly select between distinct precursor proteins. In addition, we show here for the first time that a bacterial c-type cytochrome utilizes the canonical, Sec-dependent export pathway.

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

Cited by 25 publications

References 40 publications

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity

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