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Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine
Full text linkhe 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
Determination of the primary structure of the rat mitochondrial dicarboxylate transporter via distant homologues in yeast and Caenorhabditis elegans
No full textIdentification of the human mitochondrial ATP-Mg/Pi transporter
No full textThe functions of several members of the mitochondrial transporter family found in genome sequences are unknown. At the
same time there are other transport activities observed in intact mitochondria that have yet to be associated with specific
proteins. An example is the reversible counterexchange of ATP-Mg for Pi that accounts for the net uptake or efflux of ATP-Mg,
as Pi recycles rapidly through the membrane via the phosphate carrier.
By screening human ESTs with the sequence of the human ADP/ATP carrier (AAC1) we selected clones encoding proteins
of unknown function and containing Ca
2 +
-binding EF-hand motifs in their sequences. The corresponding cDNAs (accession
numbers AJ619961, AJ619962 and AJ619963) encode three proteins (named APC1-3) with 66 –75% identical amino acids,
and with Ca
2 +
-binding motifs in their N-terminal domains and the characteristic features of the mitochondrial carrier family in
their C-terminal domains. They were overexpressed in E. coli, purified and reconstituted into liposomes. The recombinant
proteins APC1 and APC2 transported ATP-Mg, phosphate, ATP, ADP and, less effieciently, AMP in an electroneutral H
+
-
compensated counterexchange. The APC-mediated transport was inhibited by mercurials, bathophenanthroline, tannic acid and
bromocresol purple. Little inhibition was observed with carboxyatractyloside and bonkrekate (powerful inhibitors of the
AAC1). The green fluorescence (GFP) protein fused to APC1-3 was found to be targeted to mitochondria. The transport
properties of APC1 and APC2 and their targeting to mitochondria demonstrate that they are responsible for the ATP-Mg/Pi
exchange described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one
isoform is present in all the tissues investigated. By screening the human genome databases with the cDNAs of APC1, APC2
and APC3, the corresponding genes (SLC25A24, SLC25A23 and SLC25A26, respectively) were found. They were located on
three chromosomes, 1p13.3, 19p13.3 and 9q34.13; contained10exons separated by nine introns; and all the splicing junctions
occurred in the same nucleotide regions, indicating a triplication of a common ancestral gene. The main function of the APC
isoforms is probably to catalyze the net uptake or efflux of adenine nucleotides into or from the mitochondria, thus explaining
the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations
Rapamycin reduce oxidative stress in frataxin deficient yeast cells: a possibile role of mitophagy
No full textInulin‐D‐α-Tocopherol Succinate (INVITE) Nanomicelles as a Platform for Effective Intravenous Administration of Curcumin
Full text linkThe aim of this work was to evaluate the potential of INVITE-based nanomicelles, an amphiphilic polymer constituted by inulin (INU) and vitamin E (VITE), as a platform for improving the biopharmaceutical properties of hydrophobic drugs. For this purpose, curcumin was selected as a model and curcumin-INVITE nanomicelles were prepared. This drug delivery system was characterized both in vitro for what concerns the physicochemical properties, blood compatibility, and cellular uptake, and in vivo for the evaluation of the pharmacokinetic profile. It was found that these nanomicelles released curcumin in a controlled manner, and they were able to penetrate cellular membrane. Moreover, they showed an improved pharmacokinetic profile after intravenous administration. In conclusion, INVITE micelles might constitute promising nanocarriers for improving the biopharmaceutical performance of hydrophobic drugs
IDENTIFICATION AND METABOLIC SIGNIFICANCE OF YEAST MITOCHONDRIAL TRASNPORTERS VIA BACTERIAL EXPRESSION, FUNCTIONAL RECONSTITUTION INTO PHOSPHOLIPID VESCICLES AND KNOCK-OUT STRAINS ANALYSIS
No full textThe S. cerevisiae YPR011cp is a mitochondrial carrier of adenosine-5’-phosphosulfate and 3’-phospho-adenosine 5’-phosphosulfate
Full text linkThe 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 yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in deltapH formation across peroxisomal membranes
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