Conformation-Dependent Swinging of the Matrix Loop m2 of the Mitochondrial Saccharomyces cerevisiae ADP/ATP Carrier
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- 作者：Cé ; cile Dahout-Gonzalez ; Claire Ramus ; Emmanuel Philippe Dassa ; Anne-Christine Dianoux ; and Gé ; rard Brandolin
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16310 - 16320
- 全文大小：210K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
Structure-function relationships of the membrane-embedded Saccharomyces cerevisiaemitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limitedproteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside(CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to twodistinct conformers involved in the translocation process. The proteolysis approach allowed us todemonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembranespace and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible toexternally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 andLys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively. The cysteinelabeling approach was carried out on the S161C mutant of the ADP/ATP carrier. This variant of thecarrier is fully active, displaying nucleotide transport kinetic parameters and inhibitor binding propertiessimilar to that of wild-type carrier. Alkylation experiments, carried out on mitochondria with thenonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin, showed thatCys 161 is accessible from the outside in the carrier-CATR complex, whereas it is masked in the carrier-BA complex. Taken together, our results indicate that the matrix loop m2 connecting the transmembranehelices H3 to H4 intrudes to some extent into the inner mitochondrial membrane. Its participation in thetranslocation of ADP/ATP is strongly suggested, based on the finding that its accessibility to reagentsadded outside mitochondria is modified according to the conformational state of the carrier.