Perrault syndrome type 3 caused by diverse molecular defects in CLPP

Abstract

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.

Figure 1

Import and processing of PRLTS3-causing mutations in CLPP. (a) Cartoon of Human precursor CLPP (preCLPP), illustrating the relative location of intermediate CLPP (i-CLPP) and mature CLPP (m-CLPP), including the identity of the N-terminus of m-CLPP as identified by N-terminal sequencing. (b) Radiolabelled precursor protein of CLPP^T145P (lanes 1–5), CLPP^C147S (lanes 6–10), CLPP^Y229D (lanes 11–15) and wild type CLPP (lane 16–20) was imported into mitochondria isolated from HeLa cells, in the presence or absence of a membrane potential (∆ψ) as indicated. The precursor (pre) protein was processed into an intermediate (i-) and finally mature (m-) CLPP. All radiolabelled proteins were separated by 12.5% SDS-PAGE and visualised by digital autoradiography. The full-length images are presented in Supplementary Fig. S7.

Figure 2

Wild type CLPP forms heptamers and tetradecamers in vitro in the absence of human CLPX. (a) Model of Staphylococcus aureus ClpP₁₄ (SaClpP₁₄) PDB: 5C90 highlighting the position of D170 and R171, equivalent to E225 and R226 in human CLPP (here termed hE225 and hR226 respectively). (b) Assembly of wild type or mutant CLPP under native conditions. Recombinant, untagged wild type or mutant human CLPP (4 µg) was separated by Native-PAGE and visualised by staining with Coomassie Brilliant Blue (CBB) R250. The tetradecamer (CLPP₁₄) and heptamer (CLPP₇) are indicated. The oligomeric composition of wild type human CLPP (lane 2) was compared to either CLPP^ΔC (lane 1), CLPP^RA (lane 3) or CLPP^ECRC either in the absence (lane 4) or presence (lane 5) of TCEP. (c) The tetradecamer (14-mer) and heptamer (7-mer) of recombinant, untagged wild type or mutant CLPP was separated by size exclusion chromatography (SEC) using a Superdex 200 HiLoad 16/60 pg column. Elution profiles of wild type CLPP (top panel), CLPP^∆C (second panel), CLPP^RA (middle panel), CLPP^ECRC in the absence (fourth panel) or presence of TCEP (bottom panel) were measured at 280 nm (A₂₈₀). Lines indicate the peak elution volume of thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa) and conalbumin (75 kDa).

Figure 3

CLPP^Y229D displays altered oligomerisation in vitro. (a) Model showing the surface of Human ClpP₇ (PDB: 1TG6) highlighting two adjacent subunits (subunit A in blue and subunit B in pink) showing the main chain in “spaghetti”, indicating the position of the Hp residues L104 (blue) and Y138 (blue) on subunit A and Y118 (pink) and W146 (pink) on subunit B. The relative position of the residues that are mutated in Perrault syndrome (T145, C147 and Y229) and analysed in this study are indicated in yellow. (b) Assembly of wild type CLPP and CLPP Perrault mutants under native conditions. Recombinant, untagged wild type or mutant CLPP (4 µg) was separated by Native-PAGE and visualised by staining with CBB. The oligomeric composition of wild type human CLPP (lane 2) was compared to CLPP^T145P (lane 3), CLPP^C147S (lane 4) and CLPP^Y229D (lane 5). The tetradecamer (CLPP₁₄) and heptamer (CLPP₇) are indicated, as is the monomer (CLPP) and dimer (CLPP₂) of CLPP^Y229D. (c) The high order oligomeric complexes of recombinant, untagged wild type or mutant CLPP were separated by size exclusion chromatography (SEC) using a Superdex 200 HiLoad 16/60 pg column (GE Healthcare). Elution profiles of wild type CLPP (black line and top panel), CLPP^T145P (green line), CLPP^C147S (blue line and middle panel) and CLPP^Y229D (red line and bottom panel) were measured at 280 nm (A₂₈₀). Proteins from the indicated fractions were separated by SDS-PAGE and visualised by staining with CBB. Lines indicate the peak elution volume of thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa) and conalbumin (75 kDa). The full-length gels for SEC are presented in Supplementary Fig. S8.

Figure 4

CLPP^T145P displays enhanced peptidase activity. The turnover of the fluorogenic peptide substrate Suc-LY-amc (1 mM) was monitored in the presence of either wild type CLPP (black circles), CLPP^T145P (green diamonds), CLPP^C147S (blue squares) or CLPP^Y229D (red triangles). The cleavage of Suc-LY-amc was monitored by fluorescence (excitation = 380 nm, emission = 460 nm). Error bars represent the standard error of the mean (SEM) of three independent experiments.

Figure 5

CLPP^T145P and CLPP^Y229D abolish functional association with human CLPX. (a) Human CLPX-dependent turnover of FITC-casein was monitored by a change in fluorescence at 520 nm [excitation = 490 nm] in the presence of either wild type CLPP (black bar), CLPP^T145P (green bar), CLPP^C147S (blue bar) or CLPP^Y229D (red bar). The change in fluorescence at 520 nm (ΔF_{520 nm}) of FITC-casein was calculated relative to the initial fluorescence of the substrate from three independent experiments. Error bars represent SEM. (b) To monitor the turnover of FITC-casein directly, samples as described in (a) containing either wild type CLPP (lanes 1–6) or CLPP^C147S (lanes 7–12) were separated by SDS-PAGE and monitored by fluorescence (upper panel). As a loading control, the levels of CLPX and CLPP (in each reaction) were monitored by staining with CBB (lower panels). (c) To monitor the interaction between human CLPX and CLPP, human CLPX was immunoprecipitated (IP) using a specific anti-CLPX antisera, in the absence (lanes 2) or presence (lanes 7–10) of wild type or mutant human CLPP. To ensure the specificity of the co-immunoprecipitation (co-IP) the recovery of wild type and mutant human CLPP was also monitored in the absence of human CLPX (lanes 3–6). Following co-IP of human CLPP, the input (1.25%) and the eluted (33%) proteins were separated by SDS-PAGE and transferred to PVDF, before being immunodecorated with specific antisera, as indicated. (d) Quantitation of human CLPP recovered in CLPX coIP (as described in (c) in which non-specific interaction (lanes 3–6, respectively) was subtracted from the specific interaction (lanes 7–10, respectively)) of three independent experiments. Error bars represent SEM. The full-length gels for (b) are presented in Supplementary Fig. S9 and the full-length western blot for (c) are presented in Supplementary Fig. S10.