Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP

摘要

a Import of [35S]-labelled PDIP38 preprotein into mitochondria isolated from HeLa cells, in the presence or absence of a membrane potential (∆ψ) as indicated, treated with (lanes 7–12) or without (lanes 1–6) proteinase K (Prot. K). Samples were separated by 15% Tris-glycine SDS-PAGE and analysed by digital autoradiography. Full gel is shown in Supplementary Fig. 13. b Mitochondria were incubated, either in an osmotic buffer (lanes 1–4), isotonic buffer (Swelling) to rupture the outer membrane (lanes 5–8) or buffer containing Triton X-100 (lanes 9–12), in the absence (lanes 1, 5 and 9) or presence (lanes 2–4, 6–8 and 10–12) of Prot. K for the indicated time. Samples were separated by 16.5% Tricine-buffered SDS-PAGE and subjected to immunoblotting with the appropriate antisera to visualise endogenous proteins. *Non-specific cross-reactive protein recognised by PDIP38 antisera. Full immunoblots are shown in Supplementary Fig. 14. c Specific immunoprecipitation (IP) of endogenous PDIP38 from detergent-solubilised mitochondria using anti-PDIP38 polyclonal antibodies showing co-immunoprecipitation of endogenous CLPX. The input represents 50% of total mitochondrial lysate subjected to IP. Pre preimmune serum. Full immunoblot strips are shown in Supplementary Fig. 13. d Specific immunoprecipitation of endogenous CLPX from detergent-solubilised mitochondria using anti-CLPX polyclonal antibodies showing co-immunoprecipitation of endogenous PDIP38. The input represents 50% of total mitochondrial lysate subjected to IP. Full immunoblot strips are shown in Supplementary Fig. 13.