Deletion of a conserved tetrapeptide, PPGP, in P450 2C2 results in loss of enzymatic activity without a change in its cellular location.
The sequence proline-proline-glycine-proline is highly conserved in cytochrome P450 families 1 and 2, and similar proline rich sequences are found in other cytochromes P450. Since this sequence immediately follows the NH2-terminal hydrophobic membrane insertion signal, it potentially could function as a signal either for retention of cytochrome P450 in the endoplasmic reticulum or for its correct orientation in the membrane. To test this possibility, DNA sequence coding for this tetrapeptide was deleted from cytochrome P450 2C2 cDNA. Translation of the mutated mRNA in a reticulocyte cell-free system containing canine microsomal membranes resulted in the insertion of the protein into the membrane with a topology indistinguishable from that of normal cytochrome P450 2C2. The mutated protein was expressed in COS1 cells and its distribution, assayed by immunofluorescence, was similar to that of cytochrome P450 2C2. Furthermore, if a short peptide containing a potential glycosylation site was fused to the N-terminus of the mutant protein, the new hybrid protein was glycosylated in COS1 cells and the carbohydrate moiety remained sensitive to cleavage by endoglycosidase H. These results indicate that the protein was inserted and retained in the endoplasmic reticulum membrane. Pulse-chase studies showed that the mutated protein was degraded about four times as fast as cytochrome P450 2C2. In contrast to cytochrome P450 2C2, no (omega-1) hydroxylase activity was detected in COS1 cells expressing the mutated protein at similar steady-state levels as the wild-type protein. These results indicate that, although the conserved PPGP tetrapeptide is not required for cellular localization of cytochrome P450 in the endoplasmic reticulum membrane, its deletion decreases the stability of the protein and abolishes enzymatic activity.
Szczesna-Skorupa
E
E
Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign 61801.
Straub
P
P
Kemper
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006596:Hexosaminidases