Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle.
The phototaxis receptor sensory rhodopsin-I (SR-I) was genetically truncated in the COOH terminus which leads to overexpression in Halobacterium salinarium and was expressed in the presence and absence of its transducer, HtrI. Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH probe to show that proton release to the bulk phase results from the SR-I587 to S373 photoconversion, but only in the absence of transducer. The stoichiometry is 1 proton/S373 molecule formed. When SR-I is overexpressed in the presence of HtrI, the kinetics of the thermal return of S373 to SR-I587 is biphasic. A kinetic dissection indicates that overexpressed SR-I is present in two pools: one pool which generates an SR-I molecule possessing a normal (i.e., transducer-interacting) pH-independent rate of S373 decay, and a second pool which shows the pH-dependent kinetics of transducer-free S373 decay. The truncated SR-I receptor functions normally based on the following criteria: (i) Truncated SR-I restores phototaxis (attractant and repellent responses) when expressed in a strain lacking native SR-I, but containing HtrI. (ii) The absorption spectrum and the flash-induced absorption difference spectrum are indistinguishable from those of native SR-I. (iii) The rate of decay of S373 is pH-dependent in the absence of HtrI but not in the presence of HtrI. The data presented here indicate that a proton-conducting path exists between the protonated Schiff base nitrogen and the extramembranous environment in the transducer-free receptor, and transducer binding blocks this path.
Olson
K D
KD
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston 77030.
Spudich
J L
JL
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002465:Cell Movement; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology; D017353:Gene Deletion; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D019617:Halobacterium salinarum / Q000378:metabolism / Q000502:physiology*; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D025603:Sensory Rhodopsins
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D025602:Halorhodopsins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; C082504:htrI protein, Halobacterium salinarium; D001436:Bacteriorhodopsins