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. 2019 Oct 1;30(21):2681–2694. doi: 10.1091/mbc.E19-03-0166

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© 2019 Odendall et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 2: — Tgl2 is a soluble intermembrane space protein. (A) N-terminal HA-tagged Tgl2 is functional. WT or mcp2Δtgl2Δ cells were transformed with the empty plasmid pYX142 (∅). In addition, mcp2Δtgl2Δ cells were transformed with pYX142 encoding the indicated proteins under the control of the TPI promoter. Cells were grown to logarithmic phase and spotted on SD-Leu plates in a 1:5 dilution series. Plates were incubated for growth at 30°C. (B) Tgl2 is a mitochondrial protein. Whole cell lysate (WCL) and fractions corresponding to cytosol (Cyt.), light microsomal fraction (ER), and mitochondria (Mito.) of cells expressing HA-Tgl2 were analyzed by SDS–PAGE and immunodecoration with antibodies against the HA-tag, the mitochondrial protein Tom20, a marker protein for the cytosol (Bmh1), and an ER marker protein (Erv2). (C) Tgl2 is a soluble protein. Mitochondria isolated from HA-Tgl2–expressing cells (T, total) were subjected to alkaline extraction. The supernatant (S) and pellet (P) fractions were analyzed by SDS–PAGE and immunodecoration with antibodies against the indicated proteins. Tom20, an integral MOM protein; Hep1, a soluble matrix protein. (D) Tgl2 is an IMS protein. Mitochondria as in C were treated with proteinase K (PK) under different conditions. Mitochondria were kept intact, the MOM was ruptured by hypoosmolar swelling (SW), or mitochondria were lysed by the addition of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analyzed by SDS–PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a MIM protein exposed to the IMS.