A mitochondrial insertion directs substrate selection and engagement by ClpX

The force-generating AAA+ ATPase domain of protein unfoldases is specified for many substrates and other functional partners through elaboration with accessory domains. Mitochondrial homologs of the unfoldase ClpX contain an insertion within the AAA+ domain that is absent in bacterial homologs. This mitochondrial insertion (MI) maps to the substrate-encountering face of ClpX, leading us to hypothesize that the MI directs interactions of ClpX with mitochondrial substrates, the best-characterized of which is the first enzyme in heme biosynthesis, ALAS. We find that the MI is critical for both recruitment and activation of ALAS by <i>S. cerevisiae</i> ClpX. The MI was dispensable for heme-induced, adaptor-mediated degradation of ALAS by human CLPXP, but contributed to adaptor-independent recruitment of the model substrate casein for degradation. Although truncation of the MI moderately perturbed ATPase activity in both yeast and human ClpX, this effect could be uncoupled from the requirement for the MI in the efficiency of ALAS activation by targeted mutagenesis. The MI therefore can serve both to recruit a substrate to mitochondrial ClpX and to accelerate its processing by the AAA+ motor.