Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin

Abstract

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea-Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

AtGRXcp, an Arabidopsis Chloroplastic Glutaredoxin, Is Critical for Protection against Protein Oxidative Damage

“…3 and 4). In support of this hypothesis, recent studies indicate that monothiol Grxs can modulate cellular signaling events through protein-protein interactions mediated by PICOT-HD (23,26,51). Future work will be necessary to determine the target(s) of AtGRXcp and understand the complexity of ROS regulation in planta.…”

“…Yeast Grx5 encodes a mitochondrial monothiol Grx, which is required for biogenesis of iron-sulfur clusters, whereas Grx3 and Grx4 function in detoxification of cytotoxin and cell proliferation in yeast (21)(22)(23). Interestingly, bacterial Grx4, unlike other previously characterized Grxs, can serve as a substrate for thioredoxin reductase instead of NADPH/glutathione reductase (20), suggesting that those monothiol Grxs have distinct functions.…”

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

“…These five Grxs also differ in regard to their subcellular localization. Grx1 is cytosolic, Grx3 and Grx4 are nuclear (17,23), Grx5 is mitochondrial (24), and Grx2 has a dual localization in the cytosol and mitochondria (16). Grx2 stands out among other Grxs for its efficiency in transferring reducing equivalents from reduced lipoamide to oxidized glutathione (25).…”

Drosophilap53-related protein kinase is required for PI3K/TOR pathway-dependent growth

“…A yeast two-hybrid assay has shown that Bud32 interacts with IMD (Inosine Monophosphate Dehydrogenase) proteins; thus, Drosophila Prpk might participate in guanosine synthesis (Lopreiato et al, 2004). Guanosine deficiency could explain the reduction in larval growth (guanosine auxotroph); however, da>Prpk-IR larvae grown in media supplemented with 2 mg/ml of guanosine, a treatment previously used to overcome guanosine deficiency in Drosophila (O'Donnell et al, 2000), did not recover the growth phenotype (Fig.…”

“…Importantly, the growth phenotype in larval tissue is cellautonomous, ruling out indirect effects due to alterations in the larval nutrient sensor mechanism (Colombani et al, 2003). This phenotype could be explained using data from a yeast two-hybrid assay, which shows that Bud32 interacts with IMD proteins and with glutaredoxin (Grx4) (Lopreiato et al, 2004), which suggests a role for Bud32 in nucleoside biosynthesis and REDOX balance. However, Prpk-depleted larvae fed with guanosine did not recover their normal size and no obvious REDOX variations were detected using the dihydro-dichloro-fluorescein di-acetate probe in Prpkdepleted clones (data not shown), arguing against either possibility.…”