Isolation and RNA-binding analysis of NAD(+)-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD(+)-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of al l mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Anal ysis of effects of leader binding on mitochondrial translation is complicat ed by the involvement of the enzyme in mitochondrial metabolism. We have th erefore searched for an Idh altered in RNA binding, but retaining full enzy me activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe wa s partially purified and examined for the ability to bind Cox2 mRNA. Sch. p ombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own , K. lactis and S. cerevisiae COX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what d istinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two s ubunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular i n regions involved in enzyme activity, co-factor and regulator binding. Non -conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA.