The Human Mitochondrial ADP/ATP Carriers:  Kinetic Properties and Biogenesis of Wild-Type and Mutant Proteins in the Yeast S. cerevisiae

Abstract

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.

Recruitment of NF-κB into Mitochondria Is Involved in Adenine Nucleotide Translocase 1 (ANT1)-induced Apoptosis

“…Moreover, it has been suggested that the ANT2 isoform, unlike ANT1, could import glycolytic ATP into mitochondria, and a differential regulation of ANT2 gene expression has also been demonstrated (30). However, it has also been reported that there are no important differences in the transport activity or kinetic parameters of ANT isoforms (31). In addition, deletion studies of ANT1 demonstrated that apoptosis induction by ANT1 does not depend upon its function as a mitochondrial carrier (13).…”

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup

“…Mutations in a number of nuclear DNA (nDNA)-encoded mitochondrial proteins impair OXPHOS and cause cardiomyopathy (4). Recently, two case reports demonstrated homozygous solute carrier family 25, member 4 (SCL25A4) (adenine nucleotide translocator-1, ANT1) mutations (A123D; c.111+1G > A) (5,6) in patients who had cardiomyopathy and mitochondrial myopathy without the chronic progressive external ophthalmoplegia (CPEO) characteristic of certain autosomal dominant ANT1 missense mutations (L98P, A90D, D104G, A114P, and V289M) (7)(8)(9)(10).…”

The Transmembrane Prolines of the Mitochondrial ADP/ATP Carrier Are Involved in Nucleotide Binding and Transport and Its Biogenesis

“…The protocols and materials used to perform isolation of mitochondria, ADP/ATP transport, [ 3 H]ATR binding experiments, cytochrome content measurements, and protein immunostaining are described in De Marcos et al (26). For determination of the relative ScAnc2p content mitochondria were isolated from cells grown in SGal-W.…”

“…Import reactions were performed as described in Ref. 26. The samples were subjected to a 15% SDS-PAGE and the labeled proteins were visualized with the Fluorescent Image Analyzer Fujifilm FLA-500.…”