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. 2015 Dec 17;5:18375. doi: 10.1038/srep18375

Figure 2. CLPP substrate-trapping assay using isolated mitochondria.

Figure 2

(a) Cartoon presentation of P. anserina and human CLPP homologues as well as recombinant HsCLPP variants. PaCLPP is 254 amino acids and HsCLPP 277 amino acids long. Both CLPP homologues display a conserved distribution of the canonical catalytic residues Ser, His, and Asp and contain a N-terminal mitochondrial targeting sequence (MTS). Recombinant human CLPP with a C-terminal (GGGGS)2 linker followed by a 3xFLAG-6xHis-tag has a length of 315 amino acids. Catalytic inactivation of recombinant HsCLPP was achieved by mutating its catalytic serine at position 153 of the full-length HsCLPP pre-protein to alanine. (b) Lifespan of wild type (21.8 ± 0.4; n = 32), ΔPaClpP (43.7 ± 1.0; n = 58; P = 8.3E-25), ΔPaClpP/HsClpPWT-TAG (22.7 ± 0.5; n = 42; P = 3.7E-01), and ΔPaClpP/HsClpPTRAP-TAG (56.9 ± 2.9; n = 28; P = 1.9E-17) isolates at 27 °C. Data given in parentheses are mean lifespan ± s.e.m. in days. P-values were determined in comparison to the wild-type sample by two-tailed Wilcoxon rank-sum test. (c) Overview of CLPP substrate-trapping assay and proteomics work flow. (d) Venn diagram displaying overlap of identified proteins co-purifying with 3xFLAG-6xHis-tagged active (HsCLPPWT-TAG) or inactive human CLPP (HSCLPPTRAP-TAG).