Heterologous expression of limulus rhodopsin.
Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments.
Knox
Barry E
BE
Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, USA. knoxb@upstate.edu
Salcedo
Ernesto
E
Mathiesz
Katherine
K
Schaefer
Jodi
J
Chou
Wen-Hai
WH
Chadwell
Linda V
LV
Smith
W Clay
WC
Britt
Steven G
SG
Barlow
Robert B
RB
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D004594:Electrophysiology; D005123:Eye / Q000378:metabolism; D005822:Genetic Vectors; D006737:Horseshoe Crabs / Q000378:metabolism*; D007313:Insecta; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism; D017510:Protein Folding; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014981:Xenopus / Q000378:metabolism
D018076:DNA, Complementary; D018075:RNA, Complementary; D012333:RNA, Messenger; D017299:Rod Opsins; D012243:Rhodopsin