PMID:9154934

Citation	Kumagai, H, Fujiwara, T, Matsubara, H and Saeki, K (1997) Membrane localization, topology, and mutual stabilization of the rnfABC gene products in Rhodobacter capsulatus and implications for a new family of energy-coupling NADH oxidoreductases. Biochemistry 36:5509-21
Abstract	The rnf genes in Rhodobacter capsulatus are unique nitrogen fixation genes that encode potential membrane proteins (RnfA, RnfD, and RnfE) and potential iron-sulfur proteins (RnfB and RnfC). In this study, we first analyzed the localization and topology of the RnfA, RnfB, and RnfC proteins. By activity and immunoblot analysis of expression of translational fusions to Escherichia coli alkaline phosphatase, RnfA protein was shown to span the chromatophore membrane with its odd-numbered hydrophilic regions exposed to periplasm. By alkaline treatment of membrane fractions and following immunoblot analysis using antibodies against recombinant proteins expressed in E. coli, both RnfB and RnfC proteins were revealed to situate at the periphery of the chromatophore membranes. Second, mutual interaction of the Rnf proteins was analyzed by immunochemical determinations of RnfB and RnfC proteins in rnf mutants and their complemented derivatives. The contents in cellular fractions indicated that RnfB and RnfC stabilize each other and that the presence of RnfA is necessary for stable existence of both proteins. These results support a hypothesis that the Rnf products are subunits of a membrane complex. Finally, we detected homologs of rnf genes in Haemophilus influenzae and Vibrio alginolyticus by data base searches and in E. coli by cloning of a fragment of an rnfA homolog followed by a data base search. Close comparisons revealed that RnfC has potential binding sites for NADH and FMN which are similar to those found in proton-translocating NADH:quinone oxidoreductases and that RnfA, RnfD, and RnfE show similarity to subunits of sodium-translocating NADH:quinone oxidoreductases. We predict that the putative Rnf complex represents a novel family of energy-coupling NADH oxidoreductases.
Links	PubMed Online version:10.1021/bi970014q
Keywords	Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Flavin Mononucleotide/metabolism; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Molecular Sequence Data; NAD/metabolism; Nitrogen Fixation/genetics; Operon; Oxidoreductases/metabolism; Rhodobacter capsulatus/chemistry; Rhodobacter capsulatus/genetics; Sequence Homology, Amino Acid
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHOCA:RNFA	part_of	GO:0070111: organellar chromatophore	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
RHOCA:RNFA	part_of	GO:0016021: integral component of membrane	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
RHOCA:RNFA		GO:0016021: integral to membrane	ECO:0000314:		C		Figure 1B illustrates the PhoA activity of diazotrophically grown cells split into chromatophore and soluble fractions. The high PhoA activity in chromatophore fragments and low PhoA activity in soluble fragments suggests that RnfA has alternating hydrophobic and hydrophilic regions; thus, RnfA is a transmembrane protein.	complete CACAO 8538
RHOCA:RNFA		GO:0070111: organellar chromatophore	ECO:0000314:		C		Figure 1B illustrates a high amount of alkaline phosphatase activity in the chromatophore fraction of R. capsulatus cells; therefore, a high amount of RnfA activity.	complete CACAO 8693

References

See Help:References for how to manage references in GONUTS.