Figure 7. TbPam27 is required for the formation of the presequence but not for the carrier intermediate.

(A) Left: Schematic representation of the stalled presequence intermediate induced by in vivo expression of the LDH-DHFR fusion protein in presence of aminopterin (AMT). Right, upper panels: BN-PAGE analysis of the presequence intermediate in cell lines expressing HA- or myc-tagged LDH-DHFR in the background of RNAi against either TbPam27 or TbmHsp70 as indicated. Cells were grown in the absence or presence (0.5 days) of AMT as specified. (B) Left: Schematic depiction of the stalled carrier intermediate induced by the expression of the truncated mitochondrial carrier protein (MCPΔI). Right, upper panel: BN-PAGE analysis of the carrier intermediate in a cell line expressing myc-tagged MCP12ΔI in the background of RNAi against TbPam27. For all experiments digitonin-extracted mitochondrial fractions were prepared after the indicated number of days of RNAi induction and separated on a 4–13% BN-PAGE. The resultant immunoblots were probed with anti-tag antibodies. Coomassie-stained gel sections (Coom.) serve as loading controls. In (A) and (B) the right, lower panels show SDS-PAGE analysis of whole-cell extracts of the respective cell lines. Immunoblots were probed with anti-tag antibodies, to demonstrate a constant LDH-DHFR-myc/-HA or MCP12ΔI-myc expression. The two bands shown for LDH-DHFR-myc/-HA, represent the LDH-DHFR-myc/-HA portions without (lower band) or with bound AMT (higher band). Probing for TbTim17 demonstrates integrity of the TIM complex. (C) Densitometric quantification of the BN-PAGE signals to compare the amounts of LDH-DHFR-myc (as shown in A) and MCP12ΔI-myc (as shown in B) in the TbPam27-RNAi cell line found in the respective high molecular weight complexes. The levels after 0.5 days of RNAi induction were set to 100%. Error bars correspond to the standard error of the mean of three biological replicates.