Specific Interaction with Cardiolipin Triggers Functional Activation of Dynamin-Related Protein 1
Figure 6
Interaction with CL triggers Drp1 oligomerization.
A) Left panel: representative far-UV CD spectrum of Drp1 in the absence (−) or presence of LUVs composed of 80PC/20PE or 54PC/20PE/26CL (mol/mol). Right panel: thermal denaturation curves for Drp1 incubated in the absence (−) or presence of 80PC/20PE or 54PC/20PE/26CL (mol/mol) liposomes monitored by CD at 222 nm. The ellipticity values were normalized to compare Drp1 thermal denaturation rates in the absence and presence of vesicles. Drp1 and SUV concentrations were 1 µM and 1 mM, respectively. B) Rate of GTP hydrolysis was measured by using malachite green colorimetric assay as described under Materials and Methods. Increasing concentrations of Drp1 were incubated without (⧫) or with (▪) 54PC/20PE/26CL (mol/mol) liposomes (400 µM) and 1 mM GTP at 37°C. The GTPase activity of Drp1 in solution is linearly proportional to its concentration; in contrast, in presence of PC/PE/CL liposomes Drp1 exhibits sigmoidal behavior. Values are means of two to four independent experiments ±SEM. C) Gel-filtration analysis of Drp1 WT and 4KA mutant on Sephadex S200. Drp1 (1 µM) was incubated with or without GTP (1 mM) and liposomes (1 mM) in KCl buffer containing 4 mM of MgCl2 for 30 min at 37°C. Indicated samples were treated with 2% CHAPS (w/v) and applied to the column equilibrated with KCl buffer+1% CHAPS (w/v). Fractions were collected and analyzed by SDS-PAGE and immunoblotting using anti-Drp1 monoclonal antibody. Liposome lipid composition was: 28PC/20PE/52PI, 28PC/20PE/52PS, 28PC/20PE/52PG, and 54PC/20PE/26CL (CL) (mol/mol).