TKL2, a second transketolase gene of Saccharomyces cerevisiae. Cloning, sequence and deletion analysis of the gene.
Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.
Schaaff-Gerstenschläger
I
I
Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany.
Mannhaupt
G
G
Vetter
I
I
Zimmermann
F K
FK
Feldmann
H
H
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017421:Sequence Analysis; D014170:Transformation, Genetic; D014174:Transketolase / Q000737:chemistry / Q000235:genetics*
D014174:Transketolase