Self-association of Transmembrane Domain 2 (TM2), but Not TM1, in Carnitine Palmitoyltransferase 1A ROLE OF GXXXG(A) MOTIFS
Self-association of Transmembrane Domain 2 (TM2), but Not TM1, in Carnitine Palmitoyltransferase 1A ROLE OF GXXXG(A) MOTIFS
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DOI：
10.1074/jbc.m808487200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Dixon, Ann M.
中科院分区：
文献类型：
--
作者：
Jenei, Zsuzsanna A.;Borthwick, Karen;Dixon, Ann M.
肉毒碱棕榈酰转移酶1（CPT 1）控制长链脂肪酸进入线粒体基质进行β-氧化的速率，并已报告作为寡聚体存在。我们研究了全长大鼠CPT 1A（rCPT 1A）沿着酵母线粒体中表达的N端截短/缺失突变体Delta（1-82）、Delta（1-18）和Delta（19-30）的体内寡聚化。数据表明，在肝线粒体中，体内CPT 1A作为六聚体存在，但在线粒体的制备和储存期间，寡聚化的顺序迅速降低至三聚体，使得在体外分离的线粒体中观察到六聚体和三聚体的混合物。当在酵母线粒体中表达时，携带N末端不同区段（包括两个跨膜结构域的更N末端）缺失的突变体具有相同的寡聚化模式。还使用TOXCAT试验（测量大肠杆菌内膜中的TM自缔合）研究了单个rCPT 1A跨膜（TM）结构域（TM 1、TM 2）的自缔合。在TOXCAT中，对应于TM 1的序列有最小的自缔合，但TM 2有显著的自缔合。TM 2衍生的合成肽的化学交联和分析超离心显示出与在分离的线粒体中观察到的天然rCPT 1A类似的三聚体/六聚体平衡的寡聚化。因此，TM 2肽的寡聚化行为与全长蛋白的寡聚化行为之间存在相关性。rCPT 1A TM 2的计算机分子建模突出了GXXXG和GXXXA基序在TM 2六聚体形成中的有利取向。
Carnitine palmitoyltransferase 1 (CPT1) controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for beta-oxidation and has been reported to exist as an oligomer. We have investigated the in vivo oligomerization of full-length rat CPT1A (rCPT1A) along with those of the N-terminal truncation/deletion mutants Delta(1-82), Delta(1-18), and Delta(19-30) expressed in yeast mitochondria. The data indicate that in liver mitochondria in vivo CPT1A exists as a hexamer but that during preparation and storage of mitochondria the order of oligomerization is rapidly reduced to the trimer, such that a mixture of hexamer and trimer is observed in isolated mitochondria in vitro. Mutants bearing deletions of different segments of the N terminus (including the more N-terminal of the two transmembrane domains) have the same pattern of oligomerization when expressed in yeast mitochondria. The self-association of the individual rCPT1A transmembrane (TM) domains (TM1, TM2) was also studied using the TOXCAT assay (which measures TM self-association in the Escherichia coli inner membrane). There was minimal self-association of the sequence corresponding to TM1 but significant self-association of TM2 in TOXCAT. Chemical cross-linking and analytical ultracentrifugation of a TM2-derived synthetic peptide showed oligomerization with a similar trimer/hexamer equilibrium to that observed for native rCPT1A in isolated mitochondria. Therefore, there was a correlation between the oligomerization behavior of TM2 peptide and that of the full-length protein. In silico molecular modeling of rCPT1A TM2 highlighted the favorable orientation of GXXXG and GXXXA motifs in the formation of the TM2 hexamer.