Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins

Abstract

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

Molecular Identification and Functional Characterization of Arabidopsis thaliana Mitochondrial and Chloroplastic NAD+ Carrier Proteins

“…3). Their transport affinities (K m ) for NAD ϩ are lower than that of ScNDT1, and their specific activities (V max ) values are similar or higher than those displayed by most mitochondrial carriers characterized so far (16,17). However, AtNDT1 and AtNDT2 differ for their kinetic constants, AtNDT2 being more active and exhibiting a lower affinity for purine and pyrimidine nucleotides (Table 1).…”

“…Carriers belonging to the mitochondrial carrier family (MCF) represent the second group of nucleotide transporters (16,17). The most prominent member is the mitochondrial ADP/ATP carrier AAC (4), but MCF-type nucleotide transporters have also been identified in peroxisomes (7, 18), in plastids (19, 20), and in the endoplasmic reticulum of higher plants (21).…”

“…Most NTT-type carrier proteins catalyze an ATP/ADPϩP i counter-exchange mode of transport (10 -13), but several bacterial NTT proteins mediate either H ϩ /nucleotide transport or NAD ϩ /ADP counter-exchange (12,14,15). With the exception of the bacterial NAD ϩ /ADP carrier (14), all NTT proteins exhibit 12 predicted trans-membrane domains, whereas none of the NTT proteins share structural or domain similarities to members of the mitochondrial carrier family (11).Carriers belonging to the mitochondrial carrier family (MCF) represent the second group of nucleotide transporters (16,17). The most prominent member is the mitochondrial ADP/ATP carrier AAC (4), but MCF-type nucleotide transporters have also been identified in peroxisomes (7, 18), in plastids (19, 20), and in the endoplasmic reticulum of higher plants (21).…”

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

“…T he members of the mitochondrial transporter or carrier family translocate nucleotides, amino acids, inorganic ions, keto acids, and vitamins across the mitochondrial inner membrane (1,2). Uncoupling proteins, which generate heat by dissipating the proton electrochemical gradient, also belong to the family (3)(4)(5).…”

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3′,5′-Diphosphate in Human Mitochondria

“…The mitochondrial carrier family, or the solute carrier family 25 (SLC25), 3 comprises a large group of proteins that transport a variety of substrates across the inner mitochondrial membrane and, in a few cases, across other membranes (1,2). Common structural features of the mitochondrial carrier family members consist in a tripartite structure (three repeats of ϳ100 amino acids), the presence of two transmembrane ␣-helices separated by hydrophilic loops in each repeat, and the presence of a signature motif at the C terminus of the first helix in each repeat (Ref.…”