Probing in vivo Mn²⁺speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy

Abstract

Manganese is an essential transition metal that, among other functions, can act independently of proteins to either defend against or promote oxidative stress and disease. The majority of cellular manganese exists as low molecular-weight Mn 2+ complexes, and the balance between opposing “essential” and “toxic” roles is thought to be governed by the nature of the ligands coordinating Mn 2+ . Until now, it has been impossible to determine manganese speciation within intact, viable cells, but we here report that this speciation can be probed through measurements of 1 H and 31 P electron-nuclear double resonance (ENDOR) signal intensities for intracellular Mn 2+ . Application of this approach to yeast ( Saccharomyces cerevisiae ) cells, and two pairs of yeast mutants genetically engineered to enhance or suppress the accumulation of manganese or phosphates, supports an in vivo role for the orthophosphate complex of Mn 2+ in resistance to oxidative stress, thereby corroborating in vitro studies that demonstrated superoxide dismutase activity for this species.

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

“…Phosphate signaling through Pho4p is not responsible for the aerobic lethality of sod1D pho80D mutants A loss of phosphate control in S. cerevisiae through disruption of PHO80 or PHO85 renders sod1Δ cells inviable in air and unable to effectively utilize Mn for oxidative stress suppression (Reddi et al 2009;McNaughton et al 2010;Rosenfeld et al 2010). As seen in Figure 1A, Mn supplements to the growth medium only partly restore aerobic growth to a sod1Δ pho80Δ double mutant.…”

“…A number of Mn-carboxylato complexes (Archibald and Fridovich 1982b), as well as Mn-orthophosphate (Pi) (Barnese et al 2008), have been shown to exhibit superoxide scavenging activity in vitro, and the extreme radioresistance of Deinococcus radiodurans is associated with certain Mn-Pi and -peptide complexes (Daly et al 2010). Moreover, our in vivo studies in the Baker's yeast have demonstrated a role for Mn-Pi as an important backup for Cu/Zn containing SOD (SOD1) (McNaughton et al 2010).…”

“…Deletion of either pho80 or pho85 rendered sod1Δ mutants lacking Cu/Zn SOD1 inviable in air and cells seemed nearly devoid of Mn antioxidant protection (Reddi et al 2009). By ENDOR spectroscopy, Mn-Pi levels were lowered in this strain (McNaughton et al 2010), although not to a degree expected for the profound oxygen toxicity observed. Ablation of Pho80/Pho85p inhibited Mn antioxidant activity through a mechanism other than simple loss of Mn-Pi.…”

“…Using ENDOR spectroscopy to monitor Mn speciation in intact yeast cells, we observed a close correlation between the cellular level of Mn-Pi in various yeast strains and their resistance to oxygen toxicity (McNaughton et al 2010;Szuromi 2010). Of the strains tested, the most severe oxygen toxicity was observed when phosphate control was disrupted through mutations in the phosphate-sensing kinase complex Pho80p/ Pho85p (Kaneko et al 1982;Ogawa et al 2000;Wykoff and O'shea 2001;Carroll and O'shea 2002;Lee et al 2007;Wykoff et al 2007).…”

“…Total cellular phosphates (orthophosphate and polyphosphates) were measured using the molybdate reactivity method (Ames 1966;Reddi et al 2009;McNaughton et al 2010). Cells were lysed by glass bead homogenization in 500 ml of 0.1% Triton X-100.…”