Analysis of interactions with mitochondrial mRNA using mutant forms of yeast NAD(+)-specific isocitrate dehydrogenase.
Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an allosterically regulated tricarboxylic acid cycle enzyme that has been shown to bind specifically and with high affinity to 5'-untranslated regions of yeast mitochondrial mRNAs. The absence of IDH has been shown to result in reduced expression of mitochondrial translation products, leading to the suggestion that this macromolecular interaction may contribute to regulating rates of translation. The interaction with mitochondrial mRNAs also produces a dramatic inhibition of IDH catalytic activity that is specifically alleviated by AMP, the primary allosteric activator of IDH. Using mutant forms of IDH with defined catalytic or regulatory kinetic defects, we found that residue changes altering ligand binding in the catalytic site reduce the inhibitory effect of a transcript from the mitochondrial COX2 mRNA. In contrast, residue changes altering binding of allosteric regulators do not prevent inhibition by the COX2 RNA transcript but do prevent alleviation of inhibition by AMP. Results obtained using surface plasmon resonance methods suggest that the mRNA transcript may bind at the active site of IDH. Also, the presence of AMP has little effect on overall affinity but renders the binding of mRNA ineffective in catalytic inhibition of IDH. Finally, by expressing mutant forms of IDH in vivo, we determined that detrimental effects on levels of mitochondrial translation products correlate with a substantial reduction in catalytic activity. However, concomitant loss of IDH and of citrate synthase eliminates these effects, suggesting that any role of IDH in mitochondrial translation is indirect.
Anderson
Sondra L
SL
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Lin
An-Ping
AP
McAlister-Henn
Lee
L
D002384:Catalysis; D051546:Cyclooxygenase 2 / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D009243:NAD; D012313:RNA; D007521:Isocitrate Dehydrogenase; D051546:Cyclooxygenase 2