Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Zai-Rong Zhang,Sarah Perrett

doi:10.1074/jbc.m901189200

Abstract

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.

Highlights

The tripeptide glutathione (GSH)2 is abundant in the cell
A possible reason is that binding of metal ions within the Ure2 active site may inhibit catalysis. These results indicate that Ure2 may play a complementary or backup role to yeast GRX1 (yGRX1) in protection of yeast cells against oxidant and heavy metal toxicity
The GRX activity of the pellet fraction showed a time course similar to that of thioflavin T fluorescence and plateaued at a level similar to that observed for the complete sample. These results indicate that mature fibrils retain GRX activity, but the level of GRX activity in fibrils is significantly lower than in the native dimeric form

Summary

Introduction

The tripeptide glutathione (GSH)2 is abundant in the cell. It plays an important role as a reducing agent in vivo, such as in endogenous free radical scavenging, reversible protein S-glutathionylation, and the reduction of the active sites of enzymes. The results for the GRX activity of Ure2 and its mutants toward HEDS and GSH are shown in Fig. 4 and Table 1.

Results

Conclusion