Szczesna-Skorupa E, Kemper B
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
J Cell Biol. 1989 Apr;108(4):1237-43. doi: 10.1083/jcb.108.4.1237.
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.
在体外转录/翻译/转位系统中研究了兔细胞色素P450IIC2及其修饰形式[2-赖氨酸，3-精氨酸]P450IIC2插入微粒体膜的情况。在鸡输卵管微粒体膜存在的情况下合成的细胞色素P450IIC2对碱性溶液提取具有抗性，但对蛋白酶消化敏感。相反，当在膜存在的情况下合成[2-赖氨酸，3-精氨酸]-P450IIC2时，在凝胶电泳过程中观察到两种迁移较慢的新条带。用内切糖苷酶H处理后，迁移较慢的条带与在无膜情况下合成的[2-赖氨酸，3-精氨酸]P450IIC2共迁移，表明蛋白质已被糖基化。[2-赖氨酸，3-精氨酸]P450IIC2的糖基化和非糖基化形式均对蛋白酶消化和碱性溶液从膜中提取具有抗性。对于截短形式[2-赖氨酸，3-精氨酸]P450IIC2(1-55)也获得了类似结果，只是仅观察到单一的糖基化条带，这与仅剩下的一个糖基化位点一致。与先前在杂合[2-赖氨酸，3-精氨酸]P450IIC2/甲状旁腺激素蛋白中观察到的蛋白酶加工情况相反，在膜存在的情况下几乎未观察到[2-赖氨酸，3-精氨酸]P450IIC2的NH2末端肽的切割。由于杂合蛋白中的切割发生在源自[2-赖氨酸，3-精氨酸]P450IIC2的甘氨酸25之后，切割位点COOH末端的细胞色素P450序列必定降低了切割效率。这些结果表明，通常位于膜细胞质侧的细胞色素P450，当其NH2末端插入/停止转移信号的疏水核心之前有两个碱性氨基酸时，可以完全转位到腔侧。细胞色素P450先前被认为是跨膜的几个内部疏水区域中，没有一个起停止转移信号的作用。
