Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide.
The NH2-terminal sequences of cytochromes P-450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.
Department of Physiology and Biophysics, University of Illinois 61801.
E
E
Szczesna-Skorupa
N
N
Browne
D
D
Mead
B
B
Kemper
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010281:Parathyroid Hormone / Q000235:genetics; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D010281:Parathyroid Hormone; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C019056:preproparathormone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System