Figure 7: The inner membrane protein Mba1 constitutes the C2 contact site.

(a) Western blot against the mitochondrial proteins Mba1, Aco1, Oxa1, Mrpl36, Mrpl40, Mrpl20 and Mrp51 in isolated wild-type and Δmba1 mitochondria³⁰. Please note that the absence of Mba1 does neither affect the steady-state levels of proteins of the mitoribosome (Mrpl36, Mrpl40, Mrpl20 and Mrp51) nor of the membrane insertase Oxa1. (b) Translation products were radiolabelled for 20 min with [³⁵S]-methionine in isolated wild-type (wt) and Δmba1 mitochondria. Synthesized proteins were visualized by SDS–PAGE and autoradiography. The same samples were probed with Cox2-specific antibodies to visualize steady-state levels of endogenous Cox2. Cox2 is initially synthesized in the matrix as a precursor protein (preCox2), which is proteolytically matured after its insertion into the inner membrane by the Imp1 protease in the intermembrane space; mature Cox2 (mCox2) thus depends on the co-translational membrane insertion of Cox2, which is compromised in the absence of Mba1. (c) Subtomogram average of the membrane-bound mitoribosome from Δmba1 mitochondria. Essentially all density assigned to C2 disappeared. (d) Normalized difference density (red) between the structures from Δmba1 and wild-type mitochondria, isosurface-rendered at 7 s.d.’s, superposed to the subtomogram average from wild-type mitochondria (transparent grey). The difference density co-localizes with contact site C2.