Carlo M.T. Marobbio

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.

Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified. Here the identification of the mitochondrial carrier for S‐adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, (i) the Sam5p–GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM‐requiring Bio2p) on fermentable carbon sources and a petite phenotype on non‐fermentable substrates; and (iii) both phenotypes of the knock‐out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.

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

Mitochondrial carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the mitochondrial carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renström, T., and Fredriksson, R., 2006, Genomics 88, 779–790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a mitochondrial transporter for CoA and adenosine 3′,5′-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3′,5′-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intramitochondrial (deoxy)adenine nucleotides and adenosine 3′,5′-diphosphate. This is the first time that a mitochondrial carrier for CoA and adenosine 3′,5′-diphosphate has been characterized biochemically.

Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate

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.

Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization

Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent K(m) of 207 microM for TTP, 404 microM for UTP and 435 microM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaires disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

In Saccharomyces cerevisiae, α-isopropylmalate (α-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported α-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed α-IPM transporter. Transport was saturable with a half-saturation constant of 75 ± 4 μm for α-IPM and 0.31 ± 0.04 mm for β-IPM and was inhibited by the substrates of Oac1p. Though not transported, α-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, α-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an α-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with α-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant ΔOAC1ΔLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of ΔOAC1ΔLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates α-ketoisocaproate and α-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of α-IPM, probably in exchange for oxalacetate.

Perturbed mitochondrial Ca2+ signals as causes or consequences of mitophagy induction

Mitophagy is an essential process that maintains mitochondrial quality and number, thus limiting cellular degeneration. Along with apoptosis, mitophagy participates in cellular fate decisions by eliminating damaged mitochondria. A variety of mitochondrial parameters, such as structure, membrane potential, and reactive oxygen species, are key determinants in triggering the mitophagic machinery. These parameters are also important regulators of the mitochondrial capacity for calcium (Ca2+) uptake. Rapid Ca2+ accumulation in the mitochondrial matrix allows for prompt stimulation of the organelle. This process requires a close morphofunctional coupling between mitochondria and the main intracellular Ca2+ stores. In mitophagy, the role of Ca2+ remains obscure. What role does mitochondrial Ca2+ play in metabolic sensing or in mitochondrial remodeling? Is endoplasmic reticulum (ER)-Ca2+ crosstalk involved? These are some of the questions that we address in this review.

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells.

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.

The ARL2 GTPase is required for mitochondrial morphology, motility, and maintenance of ATP levels.

ARF-like 2 (ARL2) is a member of the ARF family and RAS superfamily of regulatory GTPases, predicted to be present in the last eukaryotic common ancestor, and essential in a number of model genetic systems. Though best studied as a regulator of tubulin folding, we previously demonstrated that ARL2 partially localizes to mitochondria. Here, we show that ARL2 is essential to a number of mitochondrial functions, including mitochondrial morphology, motility, and maintenance of ATP levels. We compare phenotypes resulting from ARL2 depletion and expression of dominant negative mutants and use these to demonstrate that the mitochondrial roles of ARL2 are distinct from its roles in tubulin folding. Testing of current models for ARL2 actions at mitochondria failed to support them. Rather, we found that knockdown of the ARL2 GTPase activating protein (GAP) ELMOD2 phenocopies two of three phenotypes of ARL2 siRNA, making it a likely effector for these actions. These results add new layers of complexity to ARL2 signaling, highlighting the need to deconvolve these different cell functions. We hypothesize that ARL2 plays essential roles inside mitochondria along with other cellular functions, at least in part to provide coupling of regulation between these essential cell processes.