Fig. 1.

SFXN3 localisation in HEK293 mitochondria and import pathway in yeast mitochondria. (A) Immunoblotting of soluble fraction and insoluble fractions of HEK293 mitochondria following sodium carbonate extraction (N = 3). (B) Immunoblotting of HEK293 mitochondria and mitoplasts prepared by hypotonic swelling. Mitofusin 2 is an outer mitochondrial membrane protein, Mitofilin is an intermembrane space protein, CHCHD3 is an inner mitochondrial membrane protein and TFAM is a soluble matrix protein (N = 3). (C) ³⁵S‐labelled SFXN3 was imported into isolated wild‐type HEK293 mitochondria in the presence or absence of the inner membrane potential (ΔΨ+) for the indicated time points followed by trypsin digestion to remove unimported material. Samples were run on SDS‐PAGE (N = 2). (D–F) Immunoblots of ³⁵S‐labelled SFXN3, AAC, and ATP Synthase subunit 9 (Su9‐DHFR) imported into various control and mutant yeast mitochondria. mtHSP70 was probed as a housekeeping gene at 70 kDa. Quantification of import across yeast strains is shown as the percentage of WT M+ import. Data are shown as mean percentage ± SEM (N = 3). 5% = 5% of protein loaded into all other lanes. M+ = import under the presence of membrane potential. ΔDTom70 = Delta Tom70‐depleted. Tim9ts = Tim9 temperature sensitive. Tim 22ts = Tim22 temperature sensitive. M‐ = import into mitochondria without membrane potential. Tx = mitochondria solubilised with Triton‐X100. Where different parts of the gels were assembled in a single panel, the separation between the different gels is indicated by a perpendicular line (panels D, E and F).