Fig. 5

Interaction of Mpc2 and Mpc3 with the TIM9·10 chaperone in vitro. a Cell-free reaction mixtures producing Mpc2 (upper panel) or Mpc3 (lower panel) were supplemented with detergent (Brij35) or different concentrations of recombinantly produced TIM9·10 complex. Immunoblot of the soluble (supernatant) and insoluble (pellet) fractions of the reaction mixtures. b Mpc2 and Mpc3 solubility quantification. In the presence of detergent (absence of TIM9·10), both Mpc2 and Mpc3 were largely found in the soluble fraction. In the absence of detergent and chaperone, the majority of Mpc2 and Mpc3 was found in the insoluble fraction. Increasing the concentration of TIM9·10 complex in the cell-free reaction mixture resulted in increased solubility of Mpc2 and Mpc3; n = 4–5 for Mpc2; n = 3 for Mpc3; error bars indicate standard deviation. c Structural view of the TIM9·10 complex [26, 68]. In the chaperone complex (left), Tim9 monomers are shown in dark gray and Tim10 in light gray. Altered amino acids of the mutant variants in the TIM9·10 complex [26] are shown as colored spheres. Tim10 monomer (right) and altered amino acids in the hydrophobic cleft of TIM9·10. d Immunoblot of the soluble and insoluble fractions of the cell-free reaction mixtures producing Mpc2 or Mpc3 in the absence of TIM chaperones or in the presence of wild-type TIM9·10 (TIM9·10_WT) or mutant variants of Tim10 in the TIM9·10 complex (TIM9·10_V29K, TIM9·10_F33Q, TIM9·10_M32K, TIM9·10_F70SF33Q). e Solubility quantification shows solubility of Mpc2 and Mpc3 in the presence of TIM9·10 mutant variants comparable to the reaction condition without added chaperone complex. n = 3; error bars indicate standard deviation; *** and ** indicate the significant difference with P < 0.001 and P < 0.005, respectively, in comparison with the reaction with the WT chaperone