Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA.
Cyclophilins, the intracellular receptors for the widely used immunosuppressant cyclosporin A have been found to be peptidyl-prolyl cis/trans isomerases and have been implicated in intracellular protein folding and trafficking. The Drosophila ninaA gene encodes a photoreceptor-specific cyclophilin homolog involved in rhodopsin biogenesis. ninaA mutants have a 90% reduction in the levels of Rh1 rhodopsin. To gain insight into the role of cyclophilins in vivo, we carried out a genetic screen designed to identify functionally important regions in the ninaA protein. Over 700,000 mutagenized flies were screened for a visible ninaA phenotype and 70 independent mutations in ninaA were isolated and characterized. These mutations provide a detailed dissection of the structure/function relationships in cyclophilin. We also show that mammalian cyclophilins engineered to contain missense mutations found in two temperature-sensitive ninaA alleles display temperature-sensitive prolyl cis/trans isomerase activity.
Ondek
B
B
Howard Hughes Medical Institute, La Jolla, California.
Hardy
R W
RW
Baker
E K
EK
Stamnes
M A
MA
Shieh
B H
BH
Zuker
C S
CS
D000483:Alleles; D000591:Amino Acid Isomerases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003524:Cyclosporins / Q000378:metabolism; D004330:Drosophila / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D006801:Humans; D007301:Insect Hormones / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D002352:Carrier Proteins; D003524:Cyclosporins; D029721:Drosophila Proteins; D007301:Insect Hormones; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009838:Oligodeoxyribonucleotides; C068480:ninaA protein, Drosophila; D005020:Ethyl Methanesulfonate; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase