The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an Nlumen–Ccytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane

Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate-aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor \u3b1 (PDGF\u3b1) and Transforming Growth Factor \u3b2s (TGF\u3b2s). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency

IDENTIFICATION AND FUNCTIONAL RECONSTITUTION OF THE YEAST MITOCHONDRIAL CARRIER FOR S-ADENOSYLMETHIONINE

In Saccharomyces cerevisiae, S-adenosylmethionine (SAM) is synthesized from ATP and methionine by two synthetases, Samlp and Sam2p, that are both localized exclusively in the cytosol. SAM must therefore be imported into the mitochondria, where it is required as a methyl group donor for DNA, RNA, protein and sterol methylation and as an essential cofactor in the last steps of biotin and lipoic acid biosynthesis catalyzed by biotin synthetase (Bio2p) and lipoate synthetase (Lip5p), respectively. Here we report the identification and functional characterization of the mitochondrial SAM carrier (Sam5p) encoded by YNL003c, also known as PET8. Sam5p is 284 amino acids long and has the characteristic sequence features of the mitochondrial carrier family. Sam5p was overexpressed in bacteria, purified and reconstituted into phospholipid vesicles. It transports SAM, S¬adenosylhomocysteine, and to a lesser extent, the nonphysiological structurally¬-related compounds S-adenosylcysteine and sinefungin, but none of the many other compounds tested. SAM is transported by Sam5p either by uniport or by exchange with S-adenosylhomocysteine, which is produced from SAM in methylation reactions inside mitochondria. The green fluorescent protein (GFP) fused to Sam5p was found to be targeted to mitochondria. Cells lacking the gene for this carrier (sam5Δ cells) 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. Furthermore, both phenotypes of the sam5Δ cells were restored by directing the cytosolic SAM synthetase (Sam1p) into the mitochondria

The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5'-phosphosulfate and 3'-phospho-adenosine 5'-phosphosulfate

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 condition

Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source

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

none7nononeRimessi, Alessandro; Bonora, Massimo; Marchi, Saverio; Patergnani, Simone; Marobbio, Carlo M.T.; Lasorsa, Francesco M; Pinton, Paolo*Rimessi, Alessandro; Bonora, Massimo; Marchi, Saverio; Patergnani, Simone; Marobbio, Carlo M. T.; Lasorsa, Francesco M; Pinton, Paol