Characterization and subunit structure of the ATP synthase of the halophilic archaeon Haloferax volcanii and organization of the ATP synthase genes.
The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N'-dicyclohexylcarbodiimide. The conditions for an optimally active ATP synthase have been determined. We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides. To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A1 part of the proton-translocating archaeal ATP synthase. Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon. The calculated molecular masses of the deduced gene products are 22. 0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A). The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A0 part). This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa.
Steinert
K
K
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Federal Republic of Germany.
Wagner
V
V
Kroth-Pancic
P G
PG
Bickel-Sandkötter
S
S
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001483:Base Sequence; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D016931:Halobacteriales / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D008565:Membrane Proteins; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases