Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport
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- 作者：Nicola Giangregorio ; Annamaria Tonazzi ; Lara Console ; Cesare Indiveri ; Ferdin ; o Palmieri
- 关键词：Carnitine ; Mitochondria ; Transport ; Site-directed mutagenesis ; Mitochondrial carrier
- 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
- 出版年：2010
- 出版时间：June-July 2010
- 年：2010
- 卷：1797
- 期：6-7
- 页码：839-845
- 全文大小：1433 K
文摘
The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as 3H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10 % of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15 % of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.