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. 2016 May 25;17(7):965–981. doi: 10.15252/embr.201541273

Figure 6. Mcp3 precursor physically interacts with components of the TOM complex.

Figure 6

  1. Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing, bound material was eluted with sample buffer. Proteins were analysed by SDS–PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins, the membrane was stained with Ponceau S. I, input 5% of precursor used.
  2. Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His‐Tom22 containing cells. Mitochondria were pre‐incubated with CCCP (40 μM) to halt import. After lysis with β‐dodecyl maltoside, samples were incubated with Ni‐NTA beads. After washing, bound proteins were eluted with sample buffer and analysed by SDS–PAGE and autoradiography. I, 5% of 35S‐Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni‐NTA; n.b. 2% of non‐bound material after binding to Ni‐NTA; eluate, 100% of bound material.
  3. Radiolabelled Mcp3 was incubated with organelles described in (B). After import, mitochondria were solubilized in digitonin, and the lysate was cleared by centrifugation and incubated with or without an antibody against the His‐tag (α‐His). Samples were analysed by BN‐PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti‐His antibody are indicated by arrowhead.