In vivo assembly of rhodopsin from expressed polypeptide fragments.
Rhodopsin folding and assembly were investigated by expression of five bovine opsin gene fragments separated at points corresponding to proteolytic cleavage sites in the second or third cytoplasmic regions. The CH(1-146) and CH(147-348) gene fragments encode amino acids 1-146 and 147-348 of opsin, while the TH(1-240) and TH(241-348) gene fragments encode amino acids 1-240 and 241-348, respectively. Another gene fragment, CT(147-240), encodes amino acids 147-240. All five opsin polypeptide fragments were stably produced upon expression of the corresponding gene fragments in COS-1 cells. The singly expressed polypeptide fragments failed to form a chromophore with 11-cis-retinal, whereas coexpression of two or three complementary fragments [CH(1-146) + CH(147-348), TH(1-240) + TH(241-348), or CH(1-146) + CT(147-240) + TH(241-348)] formed pigments with spectral properties similar to wild-type rhodopsin. The NH2-terminal polypeptide in these rhodopsins showed a glycosylation pattern characteristic of wild-type COS-1 cell rhodopsin and was noncovalently associated with its complementary fragment(s). Further, the CH(1-146) + CH(147-348) rhodopsin showed substantial light-dependent activation of transducin. We conclude that the functional assembly of rhodopsin is mediated by the association of at least three protein-folding domains.
Ridge
K D
KD
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, MD, USA.
Lee
S S
SS
Yao
L L
LL
D000818:Animals; D002417:Cattle; D008027:Light; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D015398:Signal Transduction; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde