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

Figure 6. Ablation of TbPam18 and TbPam16 affects growth of procyclic form but not of bloodstream form trypanosomes.

(A) Upper panels: Growth curves of uninduced (-Tet) and induced (+Tet) procyclic TbPam18, TbPam16 and Tb927.4.650 RNAi cell lines. Arrows indicate the onset of the mitochondrial protein import phenotype. Bottom panels: Immunoblot analysis of steady-state protein levels of Cox IV and TbTim17 in whole-cell extracts of the respective RNAi cell lines. EF1a serves as loading control. The positions of Cox IV precursor (P) and mature (M) forms are indicated. (B) Growth curve of the uninduced (-Tet) and induced (+Tet) blood stream form (BSF) γL262P RNAi cell lines ablating TbPam18 or TbPam16. In (A) and (B) error bars correspond to the standard deviation (n = 3). Insets show Northern blots of total RNA from uninduced or two days induced cells which were probed for the corresponding mRNAs. Ethidiumbromide-stained rRNAs serve as loading controls.

Figure 6.

Figure 6—figure supplement 1. TbPam18 and TbPam16 are not associated with the TIM complex irrespectively whether TbPam27 is present or not.

Figure 6—figure supplement 1.

(A) Top panel: Growth curves of uninduced (-Tet) and induced (+Tet) procyclic cells expressing C-terminally triple HA-tagged TbPam18 or TbPam16 in TbPam27 RNAi cells. Insets show Northern blots of total RNA from uninduced or two days induced cells which were probed for the corresponding mRNAs. Ethidiumbromide-stained rRNAs serve as loading controls. Bottom panels: Immunoblot analysis of steady-state protein levels of TbPam18-HA or TbPam16-HA in whole-cell extracts of the respective cell lines. EF1a serves as a loading control. (B) Two days induced cell lines expressing TbPam18-HA or TbPam16-HA in TbPam27 RNAi were subjected to 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 Cox IV.