The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the “gain-of-function” mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/β linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like β-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

24 40922693-file00.docx 2 25 Summary 26Polymerase δ interacting protein of 38 kDa (PDIP38) was originally identified in a yeast two hybrid 27 screen as an interacting protein of DNA polymerase delta, more than a decade ago. Since this time 28 several subcellular locations have been reported and hence its function remains controversial. Our 29 current understanding of PDIP38 function has also been hampered by a lack of detailed biochemical 30 or structural analysis of this protein. Here we show, that human PDIP38 is directed to the 31 mitochondrion, where it resides in the matrix compartment, together with its partner protein CLPX. 32 PDIP38 is a bifunctional protein, composed of two conserved domains separated by an α-helical 33 hinge region (or middle domain). The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like 34 β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-35 terminal Zinc binding domain (ZBD) of CLPX. In contrast, the C-terminal (DUF525) domain forms 36an Immunoglobin-like β-sandwich fold, which contains a highly conserved hydrophobic groove. 37Based on the physicochemical properties of this groove, we propose that PDIP38 is required for the 38 recognition (and delivery to CLPXP) of proteins bearing specific hydrophobic degrons, potentially 39 located at the termini of the target protein. Significantly, interaction with PDIP38 stabilizes the 40 steady state levels of CLPX in vivo. Consistent with these data, PDIP38 inhibits the LONM-41 mediated turnover of CLPX in vitro. Collectively, our findings shed new light on the mechanistic 42 and functional significance of PDIP38, indicating that in contrast to its initial identification as a 43 nuclear protein, PIDP38 is a bona fide mitochondrial adaptor protein for the CLPXP protease. 44 45 46