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. 2020 Feb 27;9:e52560. doi: 10.7554/eLife.52560

Figure 3. TbPam27, but not TbPam18, TbPam16 and Tb927.4.650, is associated with the TIM complex.

(A) Cell lines expressing the indicated C-terminally triple myc- or HA-tagged proteins were subjected to co-immunoprecipitation (CoIP). 5% each of crude mitochondrial fractions (Input) and unbound proteins (Unbound), as well as 100% of the final eluates (IP) were separated by SDS-PAGE. The resulting immunoblots were probed with anti-tag antibodies and antisera against TIM subunits (TbTim17 and TimRhom I) and VDAC. (B) SILAC-IP experiments of TbPam16-HA and TbPam18-HA from digitonin-extracted mitochondria enriched fractions. Volcano plots show the mean log10 ratios (induced/uninduced) of proteins that were detected in at least two of three independent biological replicates by quantitative MS, plotted against the corresponding -log10 P values (one-sided t-test). The vertical dotted lines specify the indicated enrichment factors. The horizontal dotted line indicates a t-test significance level of 0.05. Green dots represent proteins that are found in the mitochondrial proteome (Peikert et al., 2017), red dots indicate TIM subunits and pink dots represent proteins that are enriched more than three-fold and which, according to the TriTryp database (https://tritrypdb.org/tritrypdb/), are ER-localized or have a putative ER-associated function.

Figure 3—source data 1. List of proteins identified by SILAC-MS from a pull down experiment of mitochondria-enriched fractions using C-terminally myc-tagged ACAD as a bait.
elife-52560-fig3-data1.xlsx (720.6KB, xlsx)
Figure 3—source data 2. List of proteins identified by SILAC-MS from a pull down experiment of mitochondria-enriched fractions using C-terminally HA-tagged TbPam16 as a bait.
Figure 3—source data 3. List of proteins identified by SILAC-MS from a pull down experiment of mitochondria-enriched fractions using C-terminally HA-tagged TbPam18 as a bait.
elife-52560-fig3-data3.xlsx (819.2KB, xlsx)

Figure 3.

Figure 3—figure supplement 1. Global proteomic analysis confirm that TbPam27, but not TbPam18, TbPam16 or Tb927.4.650, is associated with the TIM complex.

Figure 3—figure supplement 1.

Listing of the enrichment factors of candidate proteins (bold), TIM core components and the control protein VDAC relative to the bait (or relative to TbTim17 in the last two rows) in the five indicated SILAC-IP experiments. The TbTim17, TbTim42 and TbTim13 SILAC-IPs as well as the SILAC-IPs using the stalled presequence pathway intermediate and the carrier pathway intermediate as the bait, have been published before (Harsman et al., 2016). The SILAC-IP of ACAD has been carried out in this study (Figure 3—source data 1). Dashes indicate that the particular protein has not been detected in the relative SILAC-IP.