Caracterización funcional de los transportadores mitocondriales de la familia CaMC en "Saccharomyces cerevisiae"

Abstract

The transport of metabolites through the inner mitochondrial membrane is accomplished by related proteins belonging to the initochondrial carrier superfamily (MCF). Over the last few years a number of new MCs have been identified in yeast and man, including members of a subfainily of calcium-binding mitochondrial carriers (CaMCs), with EF-hand calcium binding motifs in their N-terminal domains. Aralarl and Citrin are two closely related human CaMCs and we have recently found that they are isoforms of the mitochondrial aspanatelglutamate carrier (AGC), regulated by calcium on the extemal face of the inner mitochondrial membrane. Aralarl and Citriii are related to Agclp, a member of the CaMC subfamily in Saccharomyces cerevisiae (902 smino acids, ORF YPR02lc), although its N-terminal domain does not have EF-hand motifs. To gain insight into the physiological role of the Agclp we disrupted the AGCl gene in S. cerevisiae and studied the resulting knock-out (agclA) cells using conventional metabolic approaches as well as 1 3 n~ucle ar magnetic resonance (NMR). Out results provide evideiice that Agclp is a yeast mitochondrial transporier for aspartate and glutamate. It plays a role in nitrogen metabolism and ornithine synthesis in mitochondria, and is required for the operation of the malate-aspartate redox shuttle in S. cerevisiae, a NADH shuttle necessary for growth on acetate and fatiy acids as carbon sources. Yn1083p, another member of the CaMC subfamily, is the orthologue of hiiman SCaMCs (Short Calcium binditig Mitochondrial Carriers) whose transporier function is yet unknown, and it harboun EF-hand motifs in its N-terminal domain. Using a yeast straiii where the YNLO83w gene has been disrupted, we have shown that the mitochondrial carrier encoded by this gene is involved in a carboxyatractyloside insensitive net adenine nucleotide transport in yeast mitochondria. Thus, Yn1083p may have a transport activity similar to that of the caz*-regulated ATP-Mg/Pi carrier identified in rat liver mitochondria. Moreover, we have shown that in yeasts deticient in the three ADPIATP carrien, the disruption of YNL083w is letbal. These results provide evidence that Yn1083p is involved in the transport of adenine iiucleotides into mitochondria.