Kd. Olson et Jl. Spudich, REMOVAL OF THE TRANSDUCER PROTEIN FROM SENSORY RHODOPSIN-I EXPOSES SITES OF PROTON RELEASE AND UPTAKE DURING THE RECEPTOR PHOTOCYCLE, Biophysical journal, 65(6), 1993, pp. 2578-2585
The phototaxis receptor sensory rhodopsin-I (SR-I) was genetically tru
ncated in the COOH terminus which leads to overexpression in Halobacte
rium salinarium and was expressed in the presence and absence of its t
ransducer, HtrI. Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was u
sed as a pH probe to show that proton release to the bulk phase result
s from the SR-1587 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 the
rmal return of S373 to SR-I587 is biphasic. A kinetic dissection indic
ates that overexpressed SR-I is present in two pools: one pool which g
enerates an SR-I molecule possessing a normal (i.e., transducer-intera
cting) pH-independent rate Of S373 decay, and a second pool which show
s the pH-dependent kinetics of transducer-free S373 decay. The truncat
ed SR-I receptor functions normally based on the following criteria: (
i) Truncated SR-I restores phototaxis (attractant and repellent respon
ses) when expressed in a strain lacking native SR-I, but containing Ht
rI. (ii) The absorption spectrum and the flash-induced absorption diff
erence 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 p
roton-conducting path exists between the protonated Schiff base nitrog
en and the extramembranous environment in the transducer-free receptor
, and transducer binding blocks this path.