Biology subjects

Kardon, J. R.

Publications and source records attributed to Kardon, J. R..

4 recordsLinked to original sources

bioRxiv2026

HAX1 drives assembly and activation of the mitochondrial intermembrane space chaperone CLPB

The multicellular metazoan lineage acquired a novel chaperone in the mitochondrial intermembrane space, the AAA+ disaggregase and refoldase CLPB. Although it is not known how they function together, CLPB and the intrinsically disordered IMS protein HAX1 interact and share disease and cellular phenotypes; loss of function in either gene causes severe congenital neutropenia as well as neuropathology and causes many proteins in the IMS and its bounding membranes to become insoluble. Through biochemical reconstitution, we here find 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. A short peptide within HAX1 is necessary for direct interaction with the ankyrin domain of CLPB, but stimulation of CLPB activity requires additional elements of HAX1. Characterization of CLPB and CLPB-HAX1 oligomers indicates that 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.

biochemistry↗

bioRxiv2026

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 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.

biochemistry↗

bioRxiv2024

An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP

Heme biosynthesis is tightly coordinated such that essential heme functions including oxygen transport, respiration, and catalysis are fully supplied without overproducing toxic heme precursors and depleting cellular iron. The initial heme biosynthetic enzyme, ALA synthase (ALAS), exhibits heme-induced degradation that is dependent on the mitochondrial AAA+ protease complex CLPXP, but the mechanism for this negative feedback regulation had not been elucidated. By biochemical reconstitution, we have discovered that POLDIP2 serves as a heme-sensing adaptor protein to deliver ALAS for degradation. Similarly, loss of POLDIP2 strongly impairs ALAS turnover in cells. POLDIP2 directly recognizes heme-bound ALAS to drive assembly of the degradation complex. The C-terminal element of ALAS, truncation of which leads to a form of porphyria (XLDPP), is dispensable for interaction with POLDIP2 but necessary for degradation. Our findings establish the molecular basis for heme-induced degradation of ALAS by CLPXP, establish POLDIP2 as a substrate adaptor for CLPXP, and provide mechanistic insight into two forms of erythropoietic protoporphyria linked to CLPX and ALAS.

biochemistry↗

bioRxiv2018

Mitochondrial ClpX activates an essential metabolic enzyme through partial unfolding

Mitochondria can control the activity, quality, and lifetime of their proteins with their autonomous system of chaperones, but the signals that direct substrate-chaperone interaction and outcome are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting incorporation of cofactor. Here, we ask how unfolding by mtClpX directs activation. We identified sequence and structural features in ALAS that position mtClpX and provide a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding was limited to a subdomain extending from the mtClpX-binding site to the active site. Unfolding along this path was required for mtClpX to gate cofactor access to the ALAS active site. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and suggests how substrate-chaperone interactions can direct the outcome of remodeling.

biochemistry↗