The assembly of a-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for a-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) (20,36). This important enzyme of the tricarboxylic acid cycle has been isolated from mammalian sources (9, 27), from Escherichia coli (22), and from the yeast Saccharomyces cerevisiae (8). The eukaryotic and prokaryotic complexes have molecular weights in excess of 2 million (9, 20), with subunit stoichiometries of 12 U each of oa-ketoglutarate and dihydrolipoyl dehydrogenase (KE1 and E3), respectively, and 24 U of dihydrolipoyl transsuccinylase (KE2) (18). A description of the subunit arrangement and structure of KGDC has emerged from studies on the in vitro reassembly of native KGDC from the separated component enzymes (18,31). Such reconstitutions, carried out mainly with the E. coli complex, have been instrumental in demonstrating the existence of a high-molecular-weight core particle composed of KE2 onto which are attached dimeric units of KE1 and E3 (18,31). Knowledge of the primary structures of the components of KGDC (4, 5, 28) and the pyruvate dehydrogenase complex (PDC) (15,29,30), gained from the cloned genes, has contributed new insights about the domains involved in catalytic activity and subunit interaction (16,17,36).Despite the large body of information bearing on structure and function relationships in KGDC and PDC, little attention has been paid in the past to the mechanisms ensuring a coordinate expression of their constituent enzymes and the * Corresponding author. manner in which they interact with one another in vivo to form the functional complexes. Characterization of the genes coding for the KE1 (24), KE2 (25), and E3 (3, 26) components of yeast KGDC have made it possible to probe such questions experimentally in a eukaryotic organism. In this study we have used two approaches to examine in vivo assembly of KGDC in S. cerevisiae. The first has relied on mutants defective in the expression of KE1 or KE2 to characterize some of the intermediates in the assembly pathway. Second, we have screened a collection of respiratory-deficient pet mutants of S. cerevisiae for lesions in KGDC. These studies have led to the identification of a new gene whose product is essential for interaction of KE1 with KE2. This gene defines a new function that appears to affect a late step in the assembly of KGDC but not PDC. MATERIALS AND METHODSYeast strains and growth media. The genotypes and sources of the strains of S. cerevisiae used are given in