Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.
The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).
Lawson
M A
MA
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Zacks
D N
DN
Derguini
F
F
Nakanishi
K
K
Spudich
J L
JL
D000818:Animals; D001105:Archaea / Q000502:physiology*; D001667:Binding, Competitive; D002465:Cell Movement; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007536:Isomerism; D007700:Kinetics; D015394:Molecular Structure; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D012243:Rhodopsin / Q000378:metabolism*; D013329:Structure-Activity Relationship
D012243:Rhodopsin; D012172:Retinaldehyde