Structure-guided investigation of the mechanisms linking protein import, substrate processing, and respiration in mitochondria

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Mitochondrial targeting sequences (MTS) - N-terminal motifs which target precursor proteins to the mitochondria - are cleaved by the mitochondrial processing peptidase (MPP), a heterodimer consisting of two structurally related a and ß subunits. However, the process is still poorly understood. In metazoans, MTS processing is highly complex and is linked not only to biogenesis, but also to quality control pathways coupling protein import to the energetic status of the organelle. As evidence for this coupling, we notably observed that in vertebrates, MPP-ß shares more than 50% sequence identity with the Core 1 subunit of complex III, an essential component of the electron transport chain (ETC). While there is evidence of a chimeric complex between MPP-ß and the Core 2 subunit of complex III in cells, the significance of this interaction remains completely unknown. The goals of this research program are to elucidate the molecular mechanisms of substrate cleavage by MPP and related complexes in vertebrates and understand how the process is regulated by the energetic status of the ETC. Our general hypothesis is that while MPP-a/ß cleaves MTS from most nuclear-encoded proteins as part of their normal maturation, chimeric Core-2/MPP-ß binds and cleaves a different subset of MTS in a redox-dependent manner. To carry out this research program, we will pursue the following objectives using primarily structural and proteomics approaches. Specific objectives: 1.Elucidate the structural code of substrate cleavage by MPP-a/ß and Core-2/MPP-ß. We have established a bacterial co-expression system for these heterodimeric proteases that we will use to profile their substrate selectivity, and determine their structure by X-ray crystallography 2.Develop selective cell-permeable MPP-a/ß and Core-2/MPP-ß inhibitors. We discovered that the MTS of PINK1, a kinase involved in mitochondrial quality control, binds with high affinity to MPP-a/ß. We will use this peptide to design selective peptidomimetic inhibitors screened using a FRET-based assay to measure cleavage inhibition. 3.Characterize the distribution and activity of MPP complexes in mammalian cells. Quantitative proteomics methods will be used to characterize the distribution and activity of the two heterodimeric proteases, under basal and redox stress conditions. The structure of MPP bound to substrates, combined with their kinetics of cleavage, will provide critical clues on how this protease recognizes selectively a variety of peptide sequences, which will help predict the efficiency of cleavage for other substrates. Because these complexes are essential for survival, it is difficult to perform loss-of-function study in cells; thus, selective inhibitors will be formidable tools to characterize their function and ascertain the role of the Core-2/MPP-ß chimera. Finally, the project will enable students to become highly qualified in structural biology and proteomics methods.