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 all 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. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe 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 distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in 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.
Elzinga
S D
SD
Section for Molecular Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, BioCentrum Amsterdam, The Netherlands.
van Oosterum
K
K
Maat
C
C
Grivell
L A
LA
van der Spek
H
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics; D008928:Mitochondria / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D016601:RNA-Binding Proteins; D007521:Isocitrate Dehydrogenase