Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T.
In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal.
Paula
S
S
Department of Membrane Biochemistry, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Tittor
J
J
Oesterhelt
D
D
D000838:Anions; D001120:Arginine / Q000502:physiology*; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics*; D001692:Biological Transport; D002460:Cell Line; D002713:Chlorine / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D025602:Halorhodopsins; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013912:Threonine / Q000502:physiology*; D014633:Valine / Q000737:chemistry
D000838:Anions; D025602:Halorhodopsins; D005973:Glutamine; D013912:Threonine; D002713:Chlorine; D001436:Bacteriorhodopsins; D001120:Arginine; D014633:Valine