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Molecular & Cellular Proteomics 5:1426-1436, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Modifications of the Lipoamide-containing Mitochondrial Subproteome in a Yeast Mutant Defective in Cysteine Desulfurase^*,S

Özlem Önder^,, Heeyong Yoon^,¶, Bianca Naumann, Michael Hippler, Andrew Dancis^¶,|| and Fevzi Daldal^,**

From the Department of Biology, Plant Science Institute and ^¶ Division of Hematology-Oncology, Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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 -ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system 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. 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 synthesis.

^** To whom correspondence may be addressed. Tel.: 215-898-4394; E-mail: fdaldal{at}sas.upenn.edu

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