Fig. 6
Sub-mitochondrial NME6 distribution is typical for proteins associated with the inner membrane and matrix space. Crude mitochondria (MC) from MDA-MB-231T cells were subfractionated by a two-step protocol. First, swelling-shrinking followed by mild sonication and low speed centrifugation separates mitochondrial inner compartment (pellet MCss_lp, mainly matrix and MIM) and outer compartment (supernatant MCss_ls, mainly cristae/IMS and MOM). Second, high speed centrifugations separate from each of these two fractions the soluble components (supernatants _hs) and the membrane-bound components (pellets _hp; for details see “Materials and methods”). Fractions were evenly loaded on SDS-PAGE and analyzed by immunoblotting for NME6 and different protein markers for mitochondrial subcompartments. These include: pyruvate dehydrogenase E1 component subunit alpha (PDH-E1 α; matrix), ATP synthase subunit α (a part of the peripheral F1 subcomplex of the MIM ATP synthase, facing matrix space), optic atrophy 1 (OPA1; transmembrane MIM, facing IMS), cytochrome c (peripherally bound to MIM and soluble in IMS), adenylate kinase 2 (AK2; soluble in IMS) and voltage-dependent anion-selective channel (VDAC; MOM and MIM-MOM contact sites)