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

Figure 5. Mcp3 is imported by the TOM complex.

Figure 5

  1. Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import, mitochondria were reisolated and analysed by SDS–PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N‐terminus of Mcp3. p and m, precursor and mature forms, respectively.
  2. Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild‐type or tom70/71Δ cells were incubated with radiolabelled Mcp3 or pSu9‐DHFR (as control) for the indicated time periods. After import, mitochondria were reisolated and analysed by SDS–PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild‐type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n = 3; SD).
  3. Reduced steady‐state levels of HA‐Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS–PAGE and immunodecoration with antibodies against the HA‐tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA‐Mcp3 levels were quantified in relation to Fis1 levels, and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n = 3; SD; *, P < 0.05; two‐tailed Student's t‐test).
  4. Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild‐type and tom40‐25 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS–PAGE and autoradiography as described in (B). As a control, Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN‐PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n = 3; SD).
  5. Steady‐state levels of HA‐Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild‐type and tom40‐25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA‐tag, Tom40, and Fis1 as loading control.