The CIA targeting complex is localised in the cytosol of <i>T</i>. <i>brucei</i>.

(A) Confocal microscopy of PCF T. brucei cells expressing in-situ V5-tagged CIA components. Anti-V5 antibody (green) was used to detect the CIA proteins localized throughout the cell body. Enolase (red) was used as a cytosolic marker. DAPI (blue) stained DNA. Scalebar 1 μm. The merge displays co-localization of enolase with the V5-tagged proteins. (B) Isolation of mitochondrial fraction with digitonin. PCF trypanosomes were incubated with 0.4% (w/v) digitonin and fractions were separated by centrifugation. V5-tagged targets were visualized with anti-V5 monoclonal antibody. MtHSP70 and enolase were used as mitochondrial and cytosolic markers, respectively. P = pellet; M = mitochondrial fraction; Cyt = cytosolic fraction. All methods indicated that the proteins of the CIA targeting complex are present in the cytosol of PCF T. brucei.(C) Selective permeabilisation of whole PCF T. brucei cells with digitonin: supernatants of cells incubated with increasing amounts of digitonin were assessed by Western blot. Samples were probed against -HA (CIA components) and organelle markers: TbPLA1 (cytosolic marker); TbTAO (mitochondrial marker). The graphs represent densitometric quantifications of the Western blots for each experiment. Cells permeabilised with the highest concentration of digitonin were used as 100% release control.