A REQUIREMENT FOR MATRIX PROCESSING PEPTIDASE BUT NOT FOR MITOCHONDRIAL CHAPERONIN IN THE COVALENT ATTACHMENT OF FAD TO THE YEAST SUCCINATE-DEHYDROGENASE FLAVOPROTEIN

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerev isiae is a mitochondrial heterotetramer containing a flavoprotein subu nit with an 8 alpha-N(3)-histidyl-linked FAD cofactor. The covalent li nkage of the FAD is necessary for activity. We have developed an in vi tro assay that measures the flavinylation of the flavoprotein precurso r in mitochondrial matrix fractions. Flavoprotein modification does no t depend on translocation across a membrane, but it does require prote olytic processing by the mitochondrial processing peptidase prior to f lavin attachment. Since ATP depletion, N-ethylmaleimide, or proteinase treatments of matrix fractions inhibit flavoprotein modification, at least one additional matrix protein component appears to be required. Having previously suggested that the flavoprotein begins folding befor e FAD attachment occurs, we tested whether the mitochondrial chaperoni n, heat shock protein 60, might be necessary. Co-immunoprecipitation o f the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matr ix fractions does not inhibit FAD attachment. Nonprotein components ar e also required for flavoprotein modification. In addition to ATP, eff ector molecules such as succinate, fumarate, or malate also stimulate modification. Together, these results suggest that FAD addition is an early event in succinate dehydrogenase assembly.