Broadway Neil M, Pease Richard J, Birdsey Graeme, Shayeghi Majid, Turner Nigel A, David Saggerson E
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, U.K.
Biochem J. 2003 Feb 15;370(Pt 1):223-31. doi: 10.1042/BJ20021269.
Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88 kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.
肝微粒体组分含有一种可被丙二酰辅酶A抑制的肉碱酰基转移酶（CAT）活性。有人提出[弗雷泽、科斯托芬、普赖斯和扎米特（1999年）《欧洲生物化学学会联合会快报》446，69 - 74]，这种微粒体CAT活性是由于肉碱棕榈酰转移酶1（L - CPT1）的肝脏形式被靶向定位于内质网（ER）膜以及线粒体，可能是通过N端信号序列[科恩、吉耶拉尔特、吉拉德和普里普 - 比乌斯（2001年）《生物化学杂志》276，5403 - 5411]。瞬时转染COS - 1细胞以表达一种融合蛋白，其中增强型绿色荧光蛋白与L - CPT1的C端融合。共聚焦显微镜显示这种融合蛋白定位于线粒体，可能也定位于过氧化物酶体，但不定位于内质网。对应于截短型（氨基酸1 - 328）或全长L - CPT1的cDNA在犬胰腺微粒体存在的情况下进行转录和翻译。然而，没有证据表明CPT1真正插入到内质网膜中。通过蔗糖密度梯度离心纯化的大鼠肝微粒体组分含有一种88 kDa的蛋白（p88），它可被抗L - CPT1抗体以及L - CPT1的共价抑制剂2,4 - 二硝基苯酚 - 依托莫昔利辅酶A识别。饥饿24小时后，p88的丰度和丙二酰辅酶A抑制的CAT活性大约增加了3倍。脱氧胆酸盐从微粒体中溶解p88和丙二酰辅酶A抑制的CAT活性的程度大致相同。微粒体组分含有孔蛋白，相对于总蛋白而言，其含量与粗线粒体外膜组分中的一样丰富。得出的结论是，L - CPT不被靶向定位于内质网膜，并且微粒体组分中的丙二酰辅酶A CAT是源自线粒体的L - CPT1，可能来自膜接触位点。
