Fig. 4. Expression of LONP1 active-site variants in HEK293 cells.

a Domain architecture of the Lon protease: an N-terminal domain, a central AAA + ATPase domain and a C-terminal protease domain. The AAA + domain consists of two subdomains, Walker A and Walker B. The C-terminal protease domain contains a serine and lysine dyad in the active site. We constructed doxycycline-inducible plasmids expressing 3× FLAG-tagged wild-type LONP1 and variants carrying either a K529A or S855A exchange. K529 in the Walker A motif is essential for ATP hydrolysis whereas S855 is part of the serine protease motif in the active site. b HEK293 cell lines carrying no vector (HEK293) or pcDNA5/FRT/TO-3×FLAG-LONP1 (Lon WT, L529A, or S855A) were cultured for 6 d in the presence or absence of 1 ng/mL doxycycline. Upper panel, immunoblot analysis of total proteins from the cell lines was carried out as described in the legend to Fig. 1a. Lower panel, comparison of insoluble mitochondrial proteins prepared from the cell lines was carried out as described in the legend to Fig. 1d. Comparison of insoluble mitochondrial proteins prepared from the induced cell lines. c Comparison of the insoluble mitochondrial proteins from HEK293 cells treated with siRNAs, and HEK293 cell line expressing ATPase-deficient LONP1. Upper panel, immunoblot analysis of total proteins from the cells. Lower panel, comparison of insoluble mitochondrial proteins prepared from the cell lines. Red arrowheads indicate typical insoluble proteins specific to LONP1 knockdown cells. d Comparison of the insoluble mitochondrial proteins from HEK293 cells treated with siRNAs, and endogenous LONP1-depleted HEK293 cells expressing exogenous LONP1 and its variants. Upper panel, immunoblot analysis of total proteins from the cells. Lower panel, comparison of insoluble mitochondrial proteins prepared from the cell lines. These experiments were repeated twice and representative data are shown.