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. 2012 Sep 1;23(17):3420–3428. doi: 10.1091/mbc.E12-02-0107

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© 2012 Pusnik et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — pATOM36 is an integral protein of the mitochondrial outer membrane. (A) Immunoblots of total (Tot), digitonin-extracted crude cytosolic (Cyt), and mitochondrial (Mit) fractions from a cell line expressing pATOM36 with an HA tag at its C-terminus (pATOM36-HA) probed with anti-HA antibodies (HA). VDAC and elongation factor 1a (EF-1a) serve as mitochondrial outer membrane and cytosolic marker, respectively. (B) Immunoblot of an alkaline carbonate extraction of pATOM36-HA–containing mitochondria. Pe, pellet; Sup, supernatant. The integral membrane protein VDAC and the soluble mHSP70 serve as markers. (C) Top, immunoblot of untreated and proteinase K-treated (50 μg/ml) pATOM36-HA–containing mitochondria. Mitochondrial carrier protein 5 (MCP-5) serves as a loading control. Bottom, immunoblot of isolated mitoplasts (Mp) having a disrupted outer membrane and the intact pATOM36-HA–containing mitochondria (Mit) shown at the top. CYT C and mHSP70 serve as markers for the intermembrane space and the matrix, respectively. (D) Left, import of in vitro–translated, ³⁵S-Met–labeled pATOM36 into isolated mitochondria analyzed by BN-PAGE. Right, immunoblot of pATOM36-HA–containing mitochondrial extract separated on the same BN-PAGE. The main pATOM36 complex is indicated by the arrow. (E) Antibody shift experiments. Left, pATOM36-HA containing digitonin-lysed (+Digi) and intact mitochondria (−Digi) were incubated with polyclonal anti-HA antibodies (+HA, −antibody), separated by BN-PAGE, and analyzed by immunoblots using a monoclonal anti-HA antiserum (HA). Untreated pATOM36-HA (−HA, −antibody) complex is shown on the left as a control. Right, same as shown on the left, but SAM50-HA–expressing mitochondria were used.