Pernecky S J, Larson J R, Philpot R M, Coon M J
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109.
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2651-5. doi: 10.1073/pnas.90.7.2651.
The currently accepted model for the membrane topology of microsomal cytochrome P450 is that of a largely cytoplasmic domain bound by only one or two transmembrane segments at the NH2 terminus. However, as we have reported previously, P450 2E1 lacking the hydrophobic NH2-terminal signal peptide, like the full-length protein, is located in the inner cell membrane when expressed in Escherichia coli and is active with typical substrates. In the present study, additional variants of alcohol-inducible P450 2E1 as well as truncated forms of phenobarbital-inducible P450 2B4 were similarly expressed to determine the influence of the NH2-terminal region on the membrane-binding properties. After deletion of S1 (the NH2-terminal hydrophobic segment), or both S1 and L1 (the following hydrophilic region, expected to be lumenal or cytosolic), one-third of the resulting P450 2B4 (delta 2-20) and 2B4 (delta 2-27) remained membrane bound. Furthermore, the idea that the first two hydrophobic segments are required for attachment by a hairpin loop is not supported by the finding that after deletion of the S1, L1, and S2 segments about half of the P450 2E1 (delta 3-48) remained membrane bound. Since Na2CO3 treatment of the membrane fraction had no significant effect, the findings are apparently not attributable to a loose attachment or occlusion of the truncated proteins. The replacement of neutral amino acids by positively charged residues in positions 3 and 8 of P450 2E1 (delta 3-29) changed the amount in the cytosol from 35% to 50%, and the deletion of residues 2-20 or 2-27 from P450 2B4, which resulted in positive charges occurring in the NH2-terminal region, changed the amount in the cytosol from 27% to 67%. We conclude that alterations in the NH2-terminal region can change the location of the cytochrome from largely membranous to largely cytosolic and that the first two hydrophobic segments are not uniquely involved in membrane attachment.
目前被广泛接受的微粒体细胞色素P450膜拓扑结构模型是，其主要为胞质结构域，仅在NH2末端由一或两个跨膜片段连接。然而，正如我们之前报道的，缺乏疏水NH2末端信号肽的P450 2E1，与全长蛋白一样，在大肠杆菌中表达时位于细胞内膜，并对典型底物具有活性。在本研究中，类似地表达了酒精诱导型P450 2E1的其他变体以及苯巴比妥诱导型P450 2B4的截短形式，以确定NH2末端区域对膜结合特性的影响。在删除S1（NH2末端疏水片段）或同时删除S1和L1（随后的亲水区域，预期为腔面或胞质面）后，产生的P450 2B4（δ2 - 20）和2B4（δ2 - 27）中有三分之一仍与膜结合。此外，关于前两个疏水片段通过发夹环附着是必需的这一观点，并未得到以下发现的支持：在删除S1、L1和S2片段后，约一半的P450 2E1（δ3 - 48）仍与膜结合。由于用Na2CO3处理膜部分没有显著影响，这些发现显然不是由于截短蛋白的松散附着或封闭所致。将P450 2E1（δ3 - 29）第3和8位的中性氨基酸替换为带正电荷的残基，使胞质溶胶中的量从35%变为50%，而从P450 2B4中删除残基2 - 20或2 - 27，导致NH2末端区域出现正电荷，使胞质溶胶中的量从27%变为67%。我们得出结论，NH2末端区域的改变可使细胞色素的位置从主要位于膜上变为主要位于胞质溶胶中，并且前两个疏水片段并非唯一参与膜附着。
