Figure 1

The Drp1 80-loop functions akin to the Dyn1 PRD. (A) Top, color-coded domain organization of Drp1 relative to Dyn1. The relative positions of the Drp1-specific 80-loop and the VD are indicated. BSE; bundle signaling element, PH; pleckstrin homology domain, and GED; GTPase effector domain. Bottom, crystal structure of the Drp1ΔVD dimer (PDB ID: 4BEJ), with one monomer color-coded in correspondence to the primary structure (top panel), showing the relative positions of the VD and the loop 1 N (L1N) of the stalk. High conservation of the Drp1 and Dyn1 L1N sequence is denoted. (B) Top, color-coded 3D structure of Drp1 short GG (PDB ID: 4H1U) shown in two different orientations to highlight the relative positions of the 80-loop and residue R247. The 13 additional aa residues of the 80-loop that constitutes the ‘A-insert’ of Drp1-long, not present in the structure, is denoted. Bottom, An overlay of the Drp1 GG (green) and Dyn1 GG (gray; PDB ID: 2X2E) crystal structures in two different orientations depicting the conspicuous protuberance of the Drp1-specific 80-loop (orange) relative to the corresponding segment in Dyn1 (red). Alignment of the Drp1 80-loop sequence with the corresponding segment of Dyn1 showing conservation of the two consecutive T residues essential for the β-turn that connects the two β-strands. The position of the A-insert sequence that extends the 80-loop in Drp1-long is denoted. (C) Basal GTPase activity of Drp1 short GG relative to Drp1 long GG and Δ80-loop GG as a function of protein concentration. k_cat is the turnover number in min⁻¹. (D) Basal GTPase activities of Drp1 short GG and Drp1 long GG as a function of GTP concentration. The Michaelis constant (K_M) was determined by fitting the kinetic data to the Michaelis-Menten equation. Data shown are an average of three independent experiments ± SD. (E,F) SEC-MALS profiles of Drp1-short GG (D) and Drp1-long GG (E) (~40 kDa as monomers) each loaded at 25 μM onto a Superdex 75 10/300 GL column in the absence and presence of the transition-state analog, GDP.AlF_x. Arrow in (F) points to a relatively small dimer population. (G) Basal GTPase activities of Dyn1 GG and Dyn1 GG-PRD as a function of protein concentration. (H) Michaelis-Menten kinetics of Dyn1 GG and Dyn1 GG-PRD. (I,J) SEC-MALS profiles of Dyn1 GG (I) and Dyn1 GG-PRD (J) (~40 kDa as monomers) each loaded at 15 μM onto a Superdex 75 10/300 GL column in the absence and presence of the transition-state analog, GDP.AlF_x. (K) Confocal fluorescence images of mitochondrial morphology in Drp1 KO MEFs expressing either Myc-tagged Drp1 WT (top panels) or Δ80-loop Drp1 (bottom panels) stained for both Drp1 (green; left panels) and mitochondria (red). Insets show fragmented mitochondria in the case of Drp1 WT, or hyperfused mitochondrial networks in the case of Δ80-loop Drp1. (L) (left panel) Quantification of mitochondrial fragmentation in Drp1 KO MEFs cells expressing either Myc-tagged Drp1 WT or Δ80-loop Drp1. (middle panel) Co-localization of Drp1 WT and Δ80-loop Drp1, respectively, with mitochondria were determined from confocal fluorescence imaging using the Pearson correlation coefficient. (right panel) Representative western blots showing expression levels of Myc-tagged Drp1 WT and Δ80-loop Drp1 in transfected Drp1 KO MEFs. Empty vector-expressing cells served as negative control, and actin was used as loading control for total protein. (M) SEC profiles of Drp1 short GG in comparison to Drp1 Δ80-loop Drp1 each loaded at 6 μM onto a Superdex 75 10/300 GL column in the absence and presence of the transition-state analog, GDP.AlF_x.