Figure 3
From: Perrault syndrome type 3 caused by diverse molecular defects in CLPP
CLPPY229D 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 CLPPT145P (lane 3), CLPPC147S (lane 4) and CLPPY229D (lane 5). The tetradecamer (CLPP14) and heptamer (CLPP7) are indicated, as is the monomer (CLPP) and dimer (CLPP2) of CLPPY229D. (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), CLPPT145P (green line), CLPPC147S (blue line and middle panel) and CLPPY229D (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.
