Indiveri Cesare, Giangregorio Nicola, Iacobazzi Vito, Palmieri Ferdinando
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
Biochemistry. 2002 Jul 9;41(27):8649-56. doi: 10.1021/bi012183n.
By use of site-directed mutagenesis in combination with chemical modification of mutated proteins, the role of the six Cys residues in the transport function of the rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli, purified, and reconstituted in liposomes. The efficiency of incorporation into liposomes of the reconstituted proteins was lower for all constructs lacking Cys-23. Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria. Substitution of Cys-136 with Ser caused a nearly complete loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type CAC and the mutants containing Cys-136 showed substrate protection against NEM and MTSES inhibition and against NEM labeling. The data show that none of the native cysteines is essential for the transport mechanism and that Cys-136 is the major target of SH reagents and raise the hypothesis that Cys-136 is accessible from the external medium and is located at, or near, the substrate binding site. A model of the CAC is proposed in which the matrix hydrophilic loop containing Cys-136 protrudes into the membrane between the transmembrane domains of the protein.
通过定点诱变结合对突变蛋白的化学修饰，研究了六个半胱氨酸（Cys）残基在大鼠线粒体肉碱载体（CAC）转运功能中的作用。几个半胱氨酸残基被丝氨酸（Ser）取代的CAC突变体在大肠杆菌中过表达、纯化并重组到脂质体中。对于所有缺失Cys-23的构建体，重组蛋白掺入脂质体的效率较低。单、双和四重取代突变体的V(max)与野生型相当。根据肉碱的内部和外部转运亲和力（K(m)）值以及它们与线粒体中测量值的比较，重组CAC在脂质体中单向定向，与线粒体相比是外侧向外。用Ser取代Cys-136导致CAC对N-乙基马来酰亚胺、氢溴酸（2-氨基乙基）甲硫基磺酸盐（MTSES）和其他亲水性SH试剂几乎完全丧失敏感性，但对非常疏水的N-苯基马来酰亚胺不敏感。野生型CAC和含有Cys-136的突变体对NEM和MTSES抑制以及NEM标记表现出底物保护作用。数据表明，天然半胱氨酸残基对转运机制都不是必需的，Cys-136是SH试剂的主要作用靶点，并提出了一个假说，即Cys-136可从外部介质接近，位于底物结合位点处或附近。提出了一个CAC模型，其中包含Cys-136的基质亲水环突出到蛋白质跨膜结构域之间的膜中。
