How is mitochondrial protein activity controlled?

Mitochondria must adapt their functions to eukaryotic cellular needs, which are dynamic across changing environmental conditions, different developmental states, and the age and health of the organism. Mitochondria regulate and maintain the proteins that execute these functions with a protein unfoldase/protease machinery that is more closely related to the machinery of its bacterial endosymbiont ancestor than the 26S proteasome and other unfoldases of the eukaryotic cytoplasm. Mutation or deficiency in mitochondrial unfoldases cause diverse neurodegenerative, metabolic, and developmental disorders and drive the development of cancer, but how these unfoldases determine which proteins, at what time, to activate, remodel, repair, or target for degradation is little understood. We use biochemical and structural approaches in concert with cell biological, genetic, and proteomic tools to uncover the molecular rules that underpin these decisions and to understand how these decisions drive mitochondrial function and adaptation.

How is the outcome of protein unfolding directed?

Protein activity is determined by conformational state: proteins can be turned on or off, assembled into or extracted from complexes, or prepared for degradation by dismantling their folded structure. The AAA+ unfoldase family of enzymes apply mechanical force to other proteins to enact such changes, but we understand little about how this force is directed and limited to produce diverse outcomes. We previously discovered that the mitochondrial unfoldase ClpX activates the initial enzyme in heme biosynthesis, ALA synthase (ALAS), by executing a targeted, partial unfolding that facilitates cofactor binding. We now seek to understand the sequence, structural, and/or kinetic signals encoded in this unfoldase-substrate interaction that direct arrest and release.

How are mitochondrial protein unfoldases conditionally directed to act on their substrates?

Mitochondrial protein unfoldases select and remodel their protein substrates without direction from a conditional ubiquitin tag. Relatively few of their substrates are known, and the mechanism by which they are selected at a particular time, cellular condition, or protein state is understood for even fewer. We seek to understand how mitochondrial protein unfoldases make these decisions, which shape the activity of the mitochondrial proteome.

The work of our and several other groups indicates that mitochondrial ClpX (with its partner protease ClpP) may conditionally direct degradation of ALAS in response to heme-replete conditions, signaled by a heme-binding site in ALAS. We are investigating how this apparent feedback signal may redirect ClpX from partial unfolding/activation of ALAS to complete unfolding and degradation.

The direct role of mitochondrial ClpX in several non-heme-based diseases, the cellular phenotypes resulting from impairment of its function, and proteomic surveys suggest that ClpX acts on a broad repertoire of mitochondrial proteins. We are particularly interested in understanding how ClpX contributes to the biogenesis of several mitochondrial protein complexes and how ClpX conditionally recognizes substrates to drive and respond to changes in cell state.