Fig 2.

The 13.4L protein is a mitochondrial protein that strongly interacts with the membrane. (A) Steady-state levels of the 13.4-kDa subunit of complex I and the paralog 13.4L in several N. crassa strains determined by Western blotting. (B) The 13.4-kDa subunit of complex I is a peripheral arm component. Mitochondria from the wild-type (wt) and nuo12.3 strains were centrifuged in sucrose gradients (7.5 to 25%) upon digitonin solubilization. Fractions (labeled 1 to 11 from top to bottom) from the gradients were collected and analyzed by Western blotting with individual antisera against the complex I subunits 21.3c, 18.4, and 13.4 kDa, as well as against the complex III CORE II subunit. (C) Membrane localization of the 13.4L protein. Mitochondria (T) were incubated under alkaline conditions and resolved by centrifugation into supernatant (S) and pellet (P) fractions and subsequently analyzed by Western blotting. Mitochondria from the wild type (wt) (M) were also sonicated and separated into membrane structures (SMP) and soluble fraction (S).