Szczesna-Skorupa E, Straub P, Kemper B
Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign 61801.
Arch Biochem Biophys. 1993 Jul;304(1):170-5. doi: 10.1006/abbi.1993.1335.
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.
脯氨酸-脯氨酸-甘氨酸-脯氨酸序列在细胞色素P450家族1和2中高度保守，并且在其他细胞色素P450中也发现了类似的富含脯氨酸的序列。由于该序列紧跟在氨基末端疏水膜插入信号之后，它有可能作为一种信号，用于将细胞色素P450保留在内质网中或使其在膜中正确定向。为了验证这种可能性，从细胞色素P450 2C2 cDNA中删除了编码该四肽的DNA序列。在含有犬微粒体膜的网织红细胞无细胞系统中对突变的mRNA进行翻译，结果该蛋白质插入到膜中，其拓扑结构与正常细胞色素P450 2C2的拓扑结构无法区分。突变蛋白在COS1细胞中表达，通过免疫荧光测定其分布，与细胞色素P450 2C2的分布相似。此外，如果将一个含有潜在糖基化位点的短肽与突变蛋白的N末端融合，新的杂合蛋白在COS1细胞中被糖基化，并且碳水化合物部分对内切糖苷酶H的切割仍敏感。这些结果表明该蛋白质插入并保留在内质网膜中。脉冲追踪研究表明，突变蛋白的降解速度大约是细胞色素P450 2C2的四倍。与细胞色素P450 2C2相反，在表达与野生型蛋白处于相似稳态水平的突变蛋白的COS1细胞中未检测到（ω-1）羟化酶活性。这些结果表明，虽然保守的PPGP四肽对于细胞色素P450在内质网膜中的细胞定位不是必需的，但其缺失会降低蛋白质的稳定性并消除酶活性。
