Successful prediction of protein folding from an amino acid sequence is a challenge in computational biology. In order to reveal the geometric constraints that drive protein folding, highlight those constraints kept or missed by distinct lattices and for establishing which class of intra- and inter-secondary structure element interactions is the most relevant for the correct folding of proteins, we have calculated inter-alpha carbon distances in a set of 42 crystal structures consisting of mainly helix, sheet or mixed conformations. The inter-alpha carbon distances were also calculated in several lattice “hydrophobic-polar” models built from the same protein set. We found that helix structures are more prone to form “hydrophobic–hydrophobic” contacts than beta-sheet structures. At a distance lower than or equal to 3.8 Å (very short-range interactions), “hydrophobic–hydrophobic” contacts are almost absent in the native structures, while they are frequent in all the analyzed lattice models. At distances in-between 3.8 and 9.5 Å (short-/medium-range interactions), the best performing lattice for reproducing mainly helix structures is the body-centered-cubic lattice. If protein structures contain sheet portions, lattice performances get worse, with few exceptions observed for double-tetrahedral and body-centered-cubic lattices. Finally, we can observe that ab initio protein folding algorithms, i.e. those based on the employment of lattices and Monte Carlo simulated annealings, can be improved simply and effectively by preventing the generation of “hydrophobic–hydrophobic” contacts shorter than 3.8 Å, by monitoring the “hydrophobic–hydrophobic/polar–polar” contact ratio in short-/medium distance ranges and by using preferentially a body-centered-cubic lattice.

The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called mitochondrial carriers (MCs) [1]. The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions with different tissuespecific expression patterns, and the identification of several diseases caused by alterations of their genes [2]. Until now, 22 MC subfamilies have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes [1]. In particular two well characterized MC subfamilies are known to play a crucial role in activating the mitochondrial apoptotic pathway, the first is the subfamily of the ADP/ATP carriers and the second is the subfamily of the citrate carrier. ADP/ATP carriers catalyze the efflux of ATP from the mitochondrial matrix in exchange for cytosolic ADP and their specific inhibition can lead the permeability transition pore opening in case of oxidative stress [3]. Citrate carrier catalyses the efflux of citrate from the mitochondrial matrix in exchange for cytosolic malate and plays a key role in inflammation [4,5]. Our data together with literature data let us suppose that these two MC subfamilies are promising molecular targets for cancer treatment. In particular basing on our knowledge of MC structure, translocation mechanism and substrate specificity [6] we are evaluating neuroendocrine cancer cell resistance to old MC inhibitors and we are screening chemical libraries to develop new specific drugs to be used for viability assays.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.

The human SLC25A42 protein, ortholog of mitochondrial carrier Leu5p of S. cerevisiae, transports Coenzyme A and Adenosine 3’,5’-diphosphate G. Fiermonte, E. Paradies, S. Todisco, C.M.T. Marobbio, M.A Di Noia, and F. Palmieri Department of Pharmaco-Biology, Laboratory and Molecular Biology, University of Bari, Bari, Italy The essential cofactor Coenzyme A (CoA) is required in many intra-mitochondrial metabolic pathways. The CoA is synthesized outside the mitochondrial matrix, therefore must be transported into mitochondria. In S. cerevisiae, the mitochondrial carrier Leu5p is involved in the accumulation of CoA in the mitochondrial matrix. In fact, deletion of LEU5 (leu5) causes a reduction of mitochondrial coenzyme A (CoA) levels and growth defect on YP supplemented with glycerol or other non fermentative carbon sources. The closest relatives of Leu5p in human are SLC25A16 (37% identity) and SLC25A42 (31% identity). In this study we provide direct evidence that SLC25A42 is a novel transporter of CoA. SLC25A42 is localized in the mitochondrial inner membrane and is highly expressed in virtually all tissues. This protein was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles, and shown to transport CoA, dephospho-CoA, Adenosine 3’,5’-diphosphate (PAP), and (deoxy)adenine nucleotides with high specificity and by a counter-exchange mechanism. The expression of SLC25A42 protein in LEU5 cells fully restores the phenotype of the LEU5 strain, indicating that the main function of both proteins is probably to catalyze the entry of CoA into mitochondria in exchange for adenine nucleotides and PAP.

The S. cerevisiae YPR011c gene is located on chromosome 16 and encodes a protein of unknown function with a sequence containing the characteristic features of the mitochondrial carrier family (MCF). Until now YPR011c has been investigated only in microarray analysis of the genome-wide transcription profile of S. cerevisiae concerning YPR011c is available ([1]; website of Yeast Microarray Global Viewer (YMGV)). In the present study YPR011cp was overexpressed in Escherichia coli, purified and reconstituted into liposomes. Our results demonstrate that YPR011cp is a mitochondrial transporter for adenosine-5’-phosphosulfate (APS) and 3’-phospho-adenosine 5’-phosphosulfate (PAPS). Besides transporting APS and PAPS, recombinant and reconstituted YPR011cp also transports sulfate, phosphate, thiosulfate and pyrosulfate. YPR011cp functions almost exclusively by a counter-exchange mechanism; our transport measurements in the reconstituted system indicate that APS and PAPS may cross the mitochondrial membrane in both directions via YPR011cp in exchange with sulfate or phosphate. This is true only for APS, which is produced by the MET3p (ATP sulfurylase) that has a dual cellular localization: cytosolic and mitochondrial [2]. Having established the transport function in vitro, we investigated the physiological significance of YPR011cp in yeast cells. Upon a temperature shift from 30 to 45 °C, S. cerevisiae cells do not survive in the absence of APS and PAPS [3]. At 45°C using cells lacking YPR011c gene and other mutants we have demonstrated that both cytosolic and mitochondrial APS are crucial to support S. cerevisiae cell survival. In addition, our results strongly suggest that APS produced in mitochondria is transported from the mitochondrial matrix to the cytosol via YPR011cp under thermal stress conditions. Finally, the cellular quantification of methionine and total glutathione suggest that APS-mediated protection may be, at least in part, related to the synthesis of glutathione which is necessary for protecting cells at high temperatures [4] and for replenishing cells with sulfur metabolites. This is the first time that mitochondria are found involved in thermotolerance by mediating the transport of APS to the cytosol, which may be the basis of a signaling mechanism crucial for cell survival at higher temperatures. [1] H.C. Causton, et al. Mol. Biol. Cell 12 (2001) 323–337. [2] A. Sickmann, et al. Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13207–13212. [3] H. Jakubowski, E. Goldman, J. Bacteriol. 175 (1993) 5469–5476. [4] K. Sugiyama, et al., Biochem. J. 352 (2000) 71–78.

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.