Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

Organization and sequence of the gene for the human mitochondrial dicarboxylate carrier: evolution of the carrier family

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

Molecular Characterization of a Novel Mammalian DnaJ-Like Sec63p Homolog

We identified a human cDNA sequence encoding a polypeptide of 760 amino acids that shares 53% homology and 25.6% identity with the yeast DnaJ-like endoplasmic reticulum (ER) translocon component Sec63p. Three epitope-specific antisera revealed a protein of an apparent molecular mass of 83 kDa, both in human cell extracts and in dog pancreatic microsomes. Biochemical analyses show that it is an integral membrane protein of the rough ER, which has the DnaJ domain located in the ER lumen. The novel Sec63 protein could thus represent a key component of the mammalian ER protein translocation machinery.

A human cDNA sequence with homology to non-mammalian lysophosphatidic acid acyltransferases

A novel human homologue of Escherichia coli, yeast and plant 1-acylglycerol-3-phosphate acyltransferase has been isolated from U937 cell cDNA. Expression of the cloned sequence in 1-acylglycerol-3-phosphate acyltransferase-deficient E. coli resulted in increased incorporation of oleic acid into cellular phospholipids. Membranes made from COS7 cells transfected with the cDNA exhibited higher acyltransferase activity towards a range of donor fatty acyl-CoAs and lysophosphatidic acid. Northern-blot analysis of the cDNA sequence indicated high levels of expression in immune cells and epithelium. Rapid amplification of cDNA ends revealed differentially expressed splice variants, which suggests regulation of the enzyme by alternative splicing. This cDNA therefore represents the first described sequence of a mammalian gene homologous to non-mammalian lysophosphatidic acid acyltransferases.

CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis.

Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.