AtMSD1 activation depends on AtMTM1 participation
To investigate the dependence of Mn cofactor and AtMTM1 for AtMSD1
activation, we co-expressed AtMTM1 and AtMSD1 inySOD2 -mutant (ysod2Δ ) cells with Mn supplement(Figure 3) .
The ySOD2 activities increased clearly in WT cells under 100 μM
MnSO4 treatment (Figure 3A, lane 2) , indicating
the importance of Mn cofactor as previously reported by Luk et al.
(2003). Compared with the AtMSD1 transformation (Figure
3A, lanes 5, 6) , co-expression of AtMTM1 and AtMSD1increased AtMSD1 activities markedly in ysod2Δ cells(Figure 3A, lanes 5, 7) , reflecting AtMTM1 participated in
AtMSD1 activation. Mn supplementation had no substantial effect on
AtMSD1 activity (Figure 3A, lanes 6, 8) .
To examine the protective roles of AtMTM1 and AtMSD1 under oxidative
stress, we transformed ysod2Δ cells and challenged to methyl
viologen (MV), an oxidative-stress inducer (Figure 3B) .AtMSD1 transformation restored ysod2Δ cells to the WT
phenotype with MV treatment (Figure 3B, lanes 3, 4) , andAtMSD1 co-expressed with AtMTM1 revealed similar
protection ability (Figure 3B, lanes 5, 6) . The result implied
that a conserved effect of yMTM1 for AtMSD1 activation, as AtMTM1 for
ySOD2 activation (Supplemental Figure S4) .
The mitochondrial localization of AtMSD1 in ysod2Δ cells was
confirmed by subcellular fractionation and immunoblotting(Supplemental Figure S5A) . AtMSD1 was colocalized with a
mitochondrial marker porin (Supplemental Figure S5A, lanes 1,
3) . A cytosolic marker phosphoglycerate kinase (PGK) was used as
control (Supplemental Figure S5A, lanes 1, 2) . The
exogenous-expressed AtMSD1 activity and protein in ysod2Δ cells
were detected by in-gel SOD activity assay and immunoblotting(Supplemental Figure S5B, lanes 3, 4) , respectively. The AtMSD1
activity was confirmed with KCN treatment which inhibits ySOD1 activity(Supplemental Figure S5B, lanes 6, 7) .