title: Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex.
abstract: Cyanobacteria contain several genes, annotated ndh, whose products show sequence similarities to subunits found in complex I (NADH:ubiquinone oxidoreductase) of eubacteria and mitochondria. However, it is still unclear whether the cyanobacterial ndh gene products actually form a single large protein complex or exist as smaller independent complexes. To address this, we have constructed a strain of Synechocystis sp. PCC 6803 in which the C terminus of the NdhJ subunit was fused to an His(6) tag to aid isolation. Three major NdhJ-containing complexes were resolved by blue native polyacrylamide gel electrophoresis, with approximate apparent molecular masses of 460, 330, and 110 kDa. N-terminal sequencing and mass spectrometry revealed that the 460-kDa complex contained ten annotated ndh gene products. Detergent-induced fragmentation experiments indicated that the 460-kDa complex was composed of hydrophobic (150 kDa) and hydrophilic (110-130 kDa) modules similar to that found in the minimal form of complex I found in Escherichia coli, except that the electron input module was not conserved. The difference in size between the 460- and 330-kDa complexes is attributed to differences in the stoichiometry of the hydrophilic and hydrophobic modules in the complex, either 2:1 or 1:1, respectively. We have also detected the presence of two new Ndh subunits (slr1623 and sll1262) that are unrelated to subunits in the eubacterial complex I but which have homologues in the closely related chloroplast Ndh complex of maize (Funk, E., Schäfer, E., and Steinmüller, K. (1999) J. Plant Physiol. 154, 16-23). The presence of these additional subunits might reflect the use by the NDH-1 and Ndh complexes of a different, so far unidentified, electron input module.
authors.affiliation: Department of Biological Sciences, Wolfson Laboratories, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.
authors.forename: Peerada
authors.initials: P
authors.lastname: Prommeenate
authors.forename: Adrian M
authors.initials: AM
authors.lastname: Lennon
authors.forename: Christine
authors.initials: C
authors.lastname: Markert
authors.forename: Michael
authors.initials: M
authors.lastname: Hippler
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nixon
mesh_terms: D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D002853:Chromatography, Liquid; D019295:Computational Biology; D000458:Cyanobacteria / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D042967:Electron Transport Complex I / Q000096:biosynthesis / Q000737:chemistry*; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011808:Quinone Reductases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012984:Software
chemical_list: D003902:Detergents; D009249:NADP; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D009252:NADPH Dehydrogenase; D042967:Electron Transport Complex I