Structure-Function Relationships of the C-Terminal End of the Saccharomyces cerevisiae ADP/ATP Carrier Isoform 2

Benjamin Clémençon,Martial Rey,Anne-Christine Dianoux,Véronique Trézéguet,Guy J.-M Lauquin,Gérard Brandolin,Ludovic Pelosi

doi:10.1074/jbc.m709565200

Abstract

The adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in the cellular energy metabolism. Two regions of Anc2p from Saccharomyces cerevisiae are specifically photolabeled using a photoactivable ADP derivative; they are the central matrix loop, m2, and the C-terminal end. To get more insights into the structure-function relationships of the C-terminal region during nucleotide transport, we have developed two independent approaches. In the first we have deleted the last eight amino acids of Anc2p (Anc2pDeltaCter) and demonstrated that the C-terminal end of Anc2p plays an essential role in yeast growth on a non-fermentable carbon source. This resulted from impaired nucleotide binding properties of the Anc2pDeltaCter variant in line with conversion of ADP binding sites from high to low affinity. In the second we probed the ligand-induced conformational changes of Anc2p C-terminal end (i) by assessing its accessibility to anti-C-terminal antibodies and (ii) by measuring intrinsic fluorescence changes of an Anc2p mutant containing only one tryptophan residue located at its C-terminal end (Anc2p3Y-u). We show that the C-terminal region is no further accessible to antibodies when Anc2p binds non-transportable analogues of ADP. Besides, Trp-316 fluorescence is highly increased upon ligand binding, suggesting large conformational changes. Taken together, our results highlight the involvement of the Anc2p C-terminal region in nucleotide recognition, binding, and transport.

Highlights

Inhibitors carboxyatractyloside (CATR)4 and bongkrekic acid (BA)
Deletion of the Last Eight Amino Acids of Anc2p Severely Impairs Growth of Yeast Cells on Non-fermentable Carbon Source—Photolabeling of Anc2p in mitochondria with 2-azido3Ј-O-naphthoyl-[␤-32P]ADP resulted in the identification of two photolabeled sites located in the central matrix loop m2 and in the C-terminal end of the carrier, respectively [4]
No structural data are available so far about this region since the last five amino acids of the beef Anc1p (BAnc1p) are not resolved in the three-dimension structure [2] (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Chemicals—BA and [3H]atractyloside ([3H]ATR) were prepared as previously described [10, 11]. The pMD101 plasmid (TRP1/CEN6/ ARSH4)-containing ANC2 gene, coding for Anc2p, and its 3Јand 5Ј-flanking regions (between PstI and SalI restriction sites) was derived from pRS314 and was previously described [17]. Two mutations at position ϩ950 and ϩ951 (Phe codon replaced with Trp codon) were introduced by PCR using the recombinant plasmid pMDANC2--⌬W (derived from pMD101) coding for a Trp-less variant of Anc2p [17] as a template and the primers 5Ј-GATCTTGTTTGGTAAGAAGTGGAAATAAGACTAATCTGGCT-3Ј and 5Ј-AGCCAGATTAGTCTTATTTCCACTTCTTACCAAACAAGATC-3Ј. Isolation of the ADP/ATP Carriers—The ADP/ATP carrier protein from yeast mitochondria was isolated by chromatography on hydroxylapatite (Bio-Rad) following the method described in Brandolin et al [23]. Immunoreactivity Assays—The ability of anti-C-terminal antiserum to react with the membrane-bound carrier incubated with or without ligands was tested by ELISA, as previously described [24]. Because of the lower affinity of the carrier for nucleotides as compared with that for inhibitors, N-ADP and ADP were present at 50 or 100 ␮M final concentration, respectively, during all steps of the assay

RESULTS

Cell culture in YPLa

DISCUSSION