Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1 and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase (KGDC), pyruvate dehydrogenase (PDC) and glycine cleavage (GCS) complexes, respectively. Moreover, the increased protein spots corresponded to lipoamide deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched chain keto-acid dehydrogenase complex (BCDC). Like the lipoamide-containing forms of Kgd2, Lat1 and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast, and pave the road toward a thorough understanding of its precise role in lipoic acid synthhesis.