Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.

Molecular and Cellular Biology
B Repetto, A Tzagoloff

Abstract

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and th...Continue Reading

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