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.
IDENTIFICATION AND FUNCTIONAL RECONSTITUTION OF THE YEAST MITOCHONDRIAL CARRIER FOR S-ADENOSYLMETHIONINE
MAROBBIO, CARLO MARYA;AGRIMI, GENNARO;
2008-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
