Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.
NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.
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E Szczesna-Skorupa, B Kemper; NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.. J Cell Biol 1 April 1989; 108 (4): 1237–1243. doi: https://doi.org/10.1083/jcb.108.4.1237
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