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. 2010 Apr 19;285(25):19450–19459. doi: 10.1074/jbc.M110.109793

FIGURE 2.

FIGURE 2.

Effect of matrix-targeted Adh1 (m-Adh1) on mitochondrial zinc. Upper panel A, a schematic depicting the N-terminal fusion of the Sod2 mitochondrial targeting sequence directing Adh1 to the matrix, where upon import the Sod2 presequence is removed. Lower panel A, relative expression of c-Adh1 and m-Adh1 in both whole cell extracts and gradient-purified mitochondria relative to the respective loading controls Pgk1 and Por1. Panel B, wild-type yeast expressing either m-Adh1, or a catalytic inactive m-Adh1 mutant (T48A), show respiratory phenotypes when grown under Zn-limited conditions that are suppressed in the presence of excess Zn. Panel C, yeast lacking the plasma membrane zinc transporter, Zrt1, show an m-Adh1-induced respiratory phenotype without EDTA supplementation. Left panel D, total mitochondrial zinc from both c-Adh1- and m-Adh1-expressing cells obtained from cultures grown in synthetic complete Zn-replete medium. Right panel D, MonoS fractionation and Rhodzin-3 fluorescence (n = 3) of soluble mitochondrial lysates from either c-Adh1- or m-Adh1-expressing cultures. Left panel E, total mitochondrial zinc from both c-Adh1- and m-Adh1-expressing cells obtained from cultures grown in synthetic complete Zn-deficient medium. Right panel E, MonoS fractionation and Rhodzin-3 fluorescence (n = 3) of soluble mitochondrial lysates Zn-deficient cultures.