Genetic Dissection of the Phospholipid Hydroperoxidase Activity of Yeast Gpx3 Reveals Its Functional Importance

Abstract

Saccharomyces cerevisiae expresses multiple phospholipid hydroperoxide glutathione peroxidase (PHGPx)-like proteins in the absence of a classical glutathione peroxidase (cGPx), providing a unique system for dissecting the roles of these enzymes in vivo. The Gpx3 (Orp1/PHGpx3) protein transduces the hydroperoxide signal to the transcription factor Yap1, a function that could account for most GPX-dependent phenotypes. To test this hypothesis and ascertain what functions of Gpx3 can be shared by cGPx-like enzymes, we constructed a novel cGPx-like yeast enzyme, cGpx3. We confirmed that the "gap" sequences conserved among cGPxs but absent from aligned PHGPx sequences are the principal cause of the structural and functional differences of these enzymes. Peroxidase activity against a cGPx substrate was high in the cGpx3 construct, which was multimeric and had a peroxidase catalytic mechanism distinct from Gpx3; but cGpx3 was defective for phospholipid hydroperoxidase and signaling activities. cGpx3 did not complement the sensitivity to lipid peroxidation of a gpxDelta mutant, and the resistance to lipid peroxidation conferred by Gpx3 was independent of Yap1, establishing a functional role for Gpx3 phospholipid hydroperoxidase activity. Using the comparison between cGpx3 and Gpx3 in conjunction with other constructs to probe lipid peroxidation as a toxicity mechanism, we also ascertained that lipid peroxidation-dependent processes are a principal cause of cellular cadmium toxicity. The results demonstrate that phospholipid hydroperoxidase and Yap1-mediated signaling activities of Gpx3 have independent functional roles, although both functions depend on the absence of cGPx-like subunit interaction sites, and the results resolve more clearly the potential drivers of the differential selective evolution of GPx-like enzymes.

Oxidative protein damage causes chromium toxicity in yeast

“…However, such overlaps are not maintained across the full spectrum of antioxidant gene functions (Jamieson, 1998) and the differential effects reported here underscore this point: the specificities of the Gpx, Ogg1 and Msr proteins for repair of oxidative damage to distinct macromolecular groupsborne out by previous studies of differential activities and phenotypes associated with the corresponding deletion strains (Haracska et al, 2000;Kryukov et al, 2002;Avery et al, 2004;Willetts, 2004) -enabled us to discriminate between these groups as the candidate toxicity targets of Cr. It is emphasized that the present data refer to functions that protect against the continuous presence of Cr, a situation that may be more likely to be experienced naturally, rather than recovery after a brief Cr stress.…”

“…In yeast cells, expression of the PHGPx-like enzymes Gpx1-3 confers resistance to agents which have a lipid peroxidationdependent mode of action (Avery & Avery, 2001;Avery et al, 2004). This was exploited here to show that Cr toxicity towards S. cerevisiae does not require lipid peroxidation.…”

“…The yeast protein Gpx3 (PHGpx3/ Orp1) encodes a phospholipid hydroperoxidase, reducing phospholipid hydroperoxides (Avery & Avery, 2001;Avery et al, 2004). Expression of Gpx3 confers resistance in yeast to agents which have a lipid peroxidation-dependent mode of action ) (assays of lipid peroxidation itself do not resolve whether lipid peroxidation is a/the mode of action).…”

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

“…Structural studies on poplar GPX5 suggest that this enzyme might interact with Cd 21 and could therefore play a role as Cd 21 sink in defense against heavy metals (Koh et al 2007). Increased levels of GPX in poplar leaves exposed to high concentrations of cadmium and copper (Navrot et al 2006) and higher tolerance to cadmium of yeast cells overexpressing the GPX3 protein also support a putative role of GPXs in heavy metal tolerance (Avery et al 2004). Still, it is not clear whether this increased tolerance is due to a stimulated oxidative stress defense or to direct detoxification of Cd 21 by GPX.…”

“…PRXs, for example, can reduce peroxynitrite (Wong et al 2002;Sakamoto et al 2003) while some GPXs can reduce phospholipids and lipid hydroperoxides (Avery and Avery 2001;Herbette et al 2002;Takeda et al 2003). Several NS-GPXs have been shown to scavenge lipid or phospholipid hydroperoxides in vivo in Synechocystis (Gaber et al 2001) and yeast (Avery et al 2004). By contrast, CrGPX1 showed the highest activity with H 2 O 2 and could also reduce tBOOH and CuOOH, but not lipid hydroperoxides (Fu et al 2002).…”