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 cerevisiae is a mitochondrial heterotetramer containing a flavoprotein subunit with an 8alpha-N(3)-histidyl-linked FAD cofactor. The covalent linkage of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein precursor in mitochondrial matrix fractions. Flavoprotein modification does not depend on translocation across a membrane, but it does require proteolytic processing by the mitochondrial processing peptidase prior to flavin 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 before FAD attachment occurs, we tested whether the mitochondrial chaperonin, heat shock protein 60, might be necessary. Co-immunoprecipitation of the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matrix fractions does not inhibit FAD attachment. Nonprotein components are also required for flavoprotein modification. In addition to ATP, effector 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.
Robinson
K M
KM
Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Lemire
B D
BD
D000255:Adenosine Triphosphate / Q000378:metabolism; D018834:Chaperonin 60 / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D000096122:Mitochondrial Processing Peptidase
D000255:Adenosine Triphosphate; D018834:Chaperonin 60; D005182:Flavin-Adenine Dinucleotide; D005420:Flavoproteins; D008666:Metalloendopeptidases; D011994:Recombinant Proteins; D013385:Succinate Dehydrogenase; D000096122:Mitochondrial Processing Peptidase