Function and membrane topology of wild-type and mutated cytochrome P-450c21.
We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 --> Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21.
Hu
M C
MC
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
Hsu
L C
LC
Hsu
N C
NC
Chung
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D013006:Growth Hormone / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D007930:Leucine; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011392:Proline; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013255:Steroid 21-Hydroxylase; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D003577:Cytochrome P-450 Enzyme System; D011392:Proline; D013255:Steroid 21-Hydroxylase; D007930:Leucine