Branched-chain-amino-acid transaminases of yeast Saccharomyces cerevisiae

This chapter describes methods for the measurement of the branched-chain-amino-acid transaminase enzyme activity in either crude cell extracts of Saccharomyces cerevisiae, in isolated mitochondria, or in cytosolic fractions. Assays for both directions of the interconversion of branched-chain amino acids and α-keto acids are discussed. The chapter presents a simple method to purify the Bat proteins and determines their transaminase activity. Genetic screens for components required for the biosynthesis of branched-chain amino acids (leucine, isoleucine, and valine) in the yeast Saccharomyces cerevisiae have led to the identification of almost the complete set of genes involved in this process. The transaminase proteins termed Bat1p and Bat2p have been identified by two independent approaches. In one study, BAT1 encoding the mitochondrial isoform was identified as a high-copy suppressor of a temperature-sensitive mutant of the mitochondrial ABC transporter Atm1p. The existence of Bat2p in the yeast cytosol was realized through its specific recognition by anti-Bat1p antibodies. Cloning of the BAT1 and BAT2 genes revealed a high sequence similarity of the encoded proteins. In the second approach, Bat1p was discovered on the basis of its considerable homology to a mouse protein termed ECA39.