The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic α-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.
The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in coreexpressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.Hepatitis C virus (HCV), the major causative agent of non-A, non-B hepatitis, is estimated to infect 3% of the world's population (11). Viral infection persists in ϳ80% of infected individuals, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 13). Analyses of viral kinetics showed that HCV particles are continuously produced and cleared during chronic infection (46, 54). The lack of an efficient infectious cell culture model has prevented a comprehensive understanding of the viral life cycle. Important progress was made with the development of autonomously replicating HCV RNAs which produce nonstructural viral proteins upon transfection into hepatoma cells (7,27). Although these subgenomic replicons were recently extended to the full-length HCV genome encoding all viral proteins, no viral particle formation was observed (52).The HCV genome, a single plus-stranded RNA molecule of 9,600 nucleotides, encodes a single precursor polyprotein of about 3,000 amino acids (for reviews, see references 6 and 58). The precursor consists of N-terminal structural and C-terminal nonstructural components. During and after translation, the precursor protein undergoes an ordered series of proteolytic cleavages by viral and host proteases that generate the individual viral proteins. The functional properties of these proteins have been deduced by analogy with related RNA viruses and by cDNA expression experiments in mammalian cells (for a review, see reference 65).There are at least three viral proteins that have structural properties, including the core protein and the envelop...
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