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. 2006 Jun 20;103(27):10224–10229. doi: 10.1073/pnas.0603968103

Fig. 1.

Fig. 1.

Human AceCS2 is a soluble mitochondrial matrix protein. (A) Mitochondrial localization of human AceCS2Flag in HeLa cells. (B) Equal amounts (30 μg) of total cell extract (Total), mitochondria, light membranes (LM), and cytosolic proteins were analyzed by immunoblotting. (C) Submitochondrial localization of AceCS2Flag. (D) AceCS2Flag is a soluble protein. Mitochondria were isolated and either directly solubilized in SDS sample buffer (T, total) or extracted with sodium carbonate (Na2CO3, pH 11.5). The alkaline extract was separated into a membrane fraction (P, pellet) and a fraction containing sodium carbonate soluble proteins (S, supernatant) by ultracentrifugation. (E) The N terminus of human AceCS2 contains an amphipathic α-helix. A region of AceCS2 (amino acids 3–20) predicted by computational secondary structure analysis to form an α-helix was plotted as a helical wheel. Black, basic residues; red, hydrophobic residues; blue, neutral residues. (F) Illustration of the mitochondrial presequence and cleavage site of human AceCS2 as determined by N-terminal protein sequencing and liquid chromatography-tandem MS analysis. For details, see text. MPP, mitochondrial matrix processing peptidase.