The multicellular metazoan lineage acquired a novel chaperone in the mitochondrial intermembrane space, the AAA+ disaggregase and refoldase CLPB. Although it was not known how they function together, CLPB and the intrinsically disordered IMS protein HAX1 interact and their loss of function cause similar disease and cellular phenotypes, including congenital neutropenia, neuropathology, and increased protein insolubility in the IMS and its bounding membranes. In this study, we determined that HAX1 is a direct stimulatory cofactor of CLPB. HAX1 promotes oligomerization of CLPB into an active disaggregase and stimulates the ATPase and refoldase activities of the oligomeric complex. HAX1 shifts the predominant oligomeric state of CLPB from a dodecamer to an apparent hexamer elaborated with HAX1, suggesting that this smaller oligomer is important during the cycle of CLPB function with clients.

DeCosta A, Barrick R,  Kardon JR. A mitochondrial insertion directs substrate selection and engagement by the protein unfoldase ClpX. Journal of Biological Chemistry 2026, doi: https://doi.org/10.1016/j.jbc.2026.113539

(previously bioRxiv https://doi.org/10.64898/2026.02.23.707539)

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. We find that the MI is critical for both recruitment and activation of ALAS by S. cerevisiae 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.

*Kardon, J. R., Moroco, J. A., Engen, J. R., *Baker, T. A. (2018) Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding eLife 2020; 9:e54387 *co-corresponding author

In this manuscript, we used mutagenesis, biochemical reconstitution, and functional assays in vivo to describe a multivalent recognition and grip site in ALA synthase. Using hydrogen-deuterium exchange (in collaboration with John Engen's group, NEU) we observed that mitochondrial ClpX uses this site to initiate a specific partial unfolding of ALA synthase that exposes the active site, thus helping ALA synthase to bind its PLP cofactor and become active.

Kardon, J. R., Yien, Y. Y., Huston, N. C., Branco, D. S., Hildick-Smith, G. J., Rhee, K. Y., Paw, B. H., Baker, T. A. (2015) Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis. Cell 161(4):858-67

This paper describes our discovery of the mitochondrial unfoldase ClpX as an activator of the initial enzyme in heme biosynthesis, ALA synthase. Using genetics and metabolite profiling, we found that ClpX promotes the first step in heme biosynthesis, the synthesis of aminolevulinic acid (ALA). We additionally found that this function of ClpX is important for sustaining the high heme production necessary for red blood cell development (in collaboration with the Paw group, HMS).  Through biochemical reconstitution, we determined that ClpX directly activates ALA synthase by facilitating ALA synthase-cofactor complex formation.  

Yien, Y. Y., Ducamp, S., *van der Vorm, L. N., *Kardon, J. R.,Manceau, H., Kannengiesser, C., Bergonia, H. A., Kafina, M. D., Karim, Z., Gouya, L., Baker, T. A., Puy, H., Phillips, J. D., Nicolas, G. & Paw, B. H. (2017) Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria. PNAS114:E8045–E8052 *co-second authors

This highly collaborative study combined human genetics, cell model systems for erythropoiesis, and biochemistry to describe mitochondrial ClpX as a new causative allele in erythropoietic protoporphyria. Surprisingly, a heterozygous loss-of-function allele of ClpX results in hyperstimulated heme biosythesis due to reduced degradation of ALA synthase. This result corroborates another recent report of ClpX-dependent degradation of ALA synthase that is triggered by heme binding, suggesting a possible feedback mechanism that switches ClpX from activation to degradation of ALA synthase when heme levels are too high.