Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.

We have previously described the characterisation of an abundant mitochondrial protein (p40) that binds specifically to 5'-untranslated leaders of mitochondrial mRNAs in yeast. p40 consists of two polypeptides with M(r) of 40 and 39 kDa. Limited sequence analysis of p40 identifies it as the Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh). Both enzyme and RNA-binding activities are specifically lost in cells containing disruptions in either IDH1 or IDH2, the nuclear genes encoding the two subunits of the enzyme, thus conclusively identifying p40 as Idh and showing that both activities are dependent on the simultaneous presence of both subunits. Although we still must ascertain whether and how either function of Idh is regulated and whether the two functions are compatible or mutually exclusive, this combination of dehydrogenase activity and RNA-binding in a single protein may be part of a general regulatory circuit linking the need for mitochondrial function to mitochondrial biogenesis.

We have previously demonstrated that the yeast Krebs cycle enzyme NAD + -dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5P Puntranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2^3-fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2^3-fold reduced half-life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs. ß

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.