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The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

The amino acid sequence of the 2-oxoglutarate/malate carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions using, in the first instance, complex mixtures of oligonucleotides as primers and probes, with sequences based upon partial protein sequences of cyanogen bromide fragments of the purified protein. The protein sequence of the carrier, including the initiator methionine, is 314 amino acids long. The mature protein has a modified alpha-amino group, but the nature of this modification and the precise position of the mature N-terminal amino acid have not been ascertained, although it must lie in amino acids 1-4 of the deduced protein sequence. Comparison of the protein sequence with itself and with those of 3 other mitochondrial carrier proteins, ADP/ATP translocase, the phosphate carrier, and the uncoupling protein from brown fat, shows that all 4 proteins contain a 3-fold repeated sequence about 100 amino acids in length, and all the repeats are interrelated. This suggests that the members of this family of proteins have similar structures and mechanisms and that they have evolved from a common origin. The distribution of hydrophobic amino acids in the oxoglutarate/malate carrier supports the view that the domains are folded into similar structural motifs, possibly consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Clones of cDNA arising from a longer related transcript of the oxoglutarate/malate carrier gene have also been analyzed. They contain 271 additional nucleotides in the 3' noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)

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