Dynamic control of mitochondrial function by the protein unfoldase CLPX

Abstract Mitochondria must maintain and regulate their proteome to meet the varied needs of the cell throughout growth and development. Mitochondria employ several AAA+ family protein unfoldases as an important tool in accomplishing this control. We here focus on the AAA+ protein unfoldase CLPX, which can act on its own or unfold proteins for coupled degradation by its partner protease CLPP. Loss of CLPX is lethal in mice, mutations in CLPX and CLPP cause mitochondriopathies, and CLPXP drives cancer progression and is targeted by drugs in clinical trials in a diverse range of cancers. These phenotypes indicate the crucial contributions of CLPX and CLPP to mitochondrial physiology. The overall goal of this project is to reveal the mechanisms by which CLPX selects substrates at the appropriate time, thus tailoring their function to mitochondrial and cellular needs. Proteomic studies have linked CLPX to multiple likely substrates that control core mitochondrial processes, although only a few have been further characterized, and mechanisms for conditional substrate selection remain largely undetermined. One of the best-characterized functions for CLPX is to regulate heme biosynthesis, a crucial mitochondrial function, by acting on the first enzyme in this pathway, ALA synthase (ALAS). CLPX activates ALAS by partial unfolding to facilitate cofactor incorporation. When heme levels are high, CLPX (with its partner protease CLPP) also appears to inactivate ALAS by complete unfolding and degradation, signaled by a heme-binding site in ALAS. In preliminary results, we have biochemically reconstituted heme-induced degradation of ALAS by CLPXP, confirming the direct nature of this activity. We additionally discovered that degradation strongly depends on a heme-sensitive adaptor protein. The project proposed here will (1) elucidate the mechanism by which heme drives assembly and licensing of a degradation complex for ALAS, using our reconstituted system with equilibrium-binding and kinetic analyses of complex assembly and ALAS degradation in parallel with observations in cells. (2) We will determine how heme is directly sensed within the complex using spectroscopic and structural methods and test how the heme-responsiveness of this system is tuned to suit different cellular programs. (3) We will determine how a heme-sensitive adaptor in the CLPXP degradation complex tunes substrate selection by CLPX beyond ALAS, using a candidate-based approach and an unbiased proteomic approach in parallel. This study will reveal fundamental mechanisms for the conditional control of mitochondrial functions and will provide detailed molecular targets for the development of therapy for multiple diseases with a basis in mitochondrial function, including disorders of heme biosynthesis and cancer.