Figure 6: Domain structure and topology of ATOM complex subunits.

(a) Predicted domain structure of ATOM complex subunits drawn to scale. The predicted domains are indicated in colours. Red, predicted transmembrane domains. (b) Immunoblots of the total (T), pellet (P) and supernatant (S) fractions of carbonate-extracted mitochondria isolated from cells expressing c-Myc-tagged ATOM69 and ATOM46 performed at pH 11.5 and analysed by anti-c-Myc antiserum. VDAC and cytochrome c (Cyt c) serve as marker for an integral and peripheral membrane protein, respectively (c). Immunoblots of a protease protection assay probed for ATOM69 and ATOM46 using gradient-purified wild-type mitochondria. The intermembrane space protein Tim9 serves as a control. Bottom graph, quantification of the ratios between untreated and proteinase K-treated samples of the indicated proteins. (d) Immunoblot analysis of subcellular fractions from transgenic trypansomes expressing c-Myc-tagged variants of ATOM69 and ATOM46 that lack the predicted transmembrane domains. Whole cells (T), digitonin-extracted crude mitochondria (P) and cytosol (S) were analysed. VDAC and EF1a serve as mitochondrial or cytosolic markers, respectively. TEV, TEV protease cleavage site. Asterisks indicate the untagged versions of ATOM69 and ATOM46.