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. 2013 Apr 9;110(15):E1342-51.
doi: 10.1073/pnas.1300855110. Epub 2013 Mar 25.

Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

Sajjan Koirala  1 , Qian Guo, Raghav Kalia, Huyen T Bui, Debra M Eckert, Adam Frost, Janet M Shaw
Affiliations

Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

Sajjan Koirala et al. Proc Natl Acad Sci U S A. .

Abstract

Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Fis1 is dispensable for mitochondrial fission. (A) Domain structure of WT Mdv1 and Mdv1 constructs fused to the N-terminal transmembrane anchor of yeast T20. NTE, CC, and predicted β domains are shown. (B) Quantification of mitochondrial morphology in cells expressing the indicated fission proteins. All values are mean ± SEM; n ≥ 300. Representative images of WT and fission mutant mitochondria scored are shown in Fig. S1. JSY strains 8614, 9234, 9801, 9802, and 9803 were used.
Fig. 2.
Fig. 2.
Dnm1 fission complexes assemble on mitochondria in the absence of Fis1. (A) Representative images of GFP-Dnm1 puncta on mitochondria. Differential interference contrast (DIC), GFP-Dnm1, and merged mito-RFP (mitochondrial matrix-targeted dsRed) images are shown. (Scale bar, 5 µm.) (B) Quantification of the number of cells in a population containing punctate GFP-Dnm1 fission complexes on mitochondria. All values are mean ± SEM; n ≥ 300. (C) Box-and-whisker plots showing the number of mitochondrial GFP-Dnm1 puncta per cell in the indicated strains. n ≥ 10 cells. The y axes for B and C are the same. JSY strains 9493, 9548, 9804, 9805, and 9806 were used.
Fig. 3.
Fig. 3.
Mitochondrial fission and fusion events in cells lacking Fis1. (A) Box-and-whisker plot showing the distribution of fission (n ≥ 20 cells) and fusion (n ≥ 20 cells) events in WT and strains expressing the indicated fission proteins. Total fission or fusion events per cell for a 15-min interval are indicated. (B) Representative mito-RFP–labeled mitochondria are shown undergoing fission (Upper, arrow) and fusion (Lower, arrow) in cells expressing Dnm1 and tethered Mdv1 (T20-β). (Scale bar, 1 µm.)
Fig. 4.
Fig. 4.
Mammalian Drp1 and mitochondrial-tethered adaptor proteins rescue mitochondrial fission defects in yeast. (A) Domain structures of hFis1 and Mff fused to the C-terminal outer membrane anchor of yeast Fis1 (yTM). Human MiD49 or MiD51 were targeted to the mitochondrial outer membrane via fusion to the N-terminal transmembrane anchor of yeast T20. (B) Quantification of mitochondrial morphology in cells expressing Mff plus the indicated fission proteins and mito-OMGFP. All values are mean ± SEM; n ≥ 300. (C) Quantification of mitochondrial morphologies observed in cells during induction of Drp1 and MiD51 from the MET25 promoter. The merged DIC and mito-OMGFP images to the right of the graph are representative of the mitochondrial morphology categories scored. (Scale bar, 5 µm.) (D) Time-lapse images of GFP-Drp1 at fission sites in cells expressing Mff (Top), MiD49 (Middle), or MiD51 (Bottom). Boxed regions mark fission sites imaged at 30-s intervals in each row. (Scale bar, 2 µm.)
Fig. 5.
Fig. 5.
Effects of Mff and MiD49 on Drp1 GTPase activity. (A) Time course of GTP hydrolysis by Drp1 (0.6 μM) measured in 100 μM GTP, 37 °C at high (500 mM KCl) and low (50 mM KCl) ionic strength. (B) Steady-state kinetics of Drp1 (0.6 μM) GTP hydrolysis measured at low ionic strength (50 mM KCl), 37 °C. (C) A Coomasie blue-stained gel showing velocity sedimentation of Drp1 at high and low ionic strength. P, pellet; S, supernatant; T, total. (D) Drp1 kinetic parameters determined as described in B and Materials and Methods. kcat, turnover number; Vmax, maximal rate of hydrolysis; K0.5, substrate concentration at which velocity is one-half maximal. (E and F) GTP hydrolysis by Drp1 (0.1 μM) measured in 200 μM GTP and 50 mM KCl at 37 °C in the presence and absence of the indicated adaptor proteins. Cytoplasmic domains of Mff (E) or MiD49 (F) purified from yeast were included at 0.5 μM.
Fig. 6.
Fig. 6.
Drp1 self-assembly induces lipid tubulation and constriction in vitro. (A–F) Transmission electron micrographs of negatively stained Drp1 assemblies. (A) Drp1 protomers do not assemble in the absence of nucleotide at 4 °C. (B) Drp1 assembles into limited rings in the presence of GMP-PCP at low temperature (white arrow). (C) At 25 °C, Drp1 forms spirals or stacks of rings in the presence of GMP-PCP that exclude liposomes containing molar 37% PS (asterisk). (D) Drp1 assembles around liposomes containing molar 100% PS in the presence of GMP-PCP at 25 °C. (E and F) Drp1-decorated lipid tubes assembled in the presence of GMP-PCP were imaged after treatment with 1 mM GTP for 10 s (E) or 30 min (F). (G) Average external diameters of Drp1 structures as indicated by white arrowheads in C–F. For all measurements, n = 50. (Scale bars, 50 nm.)
Fig. 7.
Fig. 7.
MiD49 copolymerizes with Drp1 and decreases polymer diameter. (A) MiD49∆TM (lacking the transmembrane domain) cosediments with Drp1. (B) liposomes containing DO DGS-NTA(Ni) (Upper) decorated with His-tagged MiD49∆TM promote flotation of Drp1 in a sucrose step gradient. Charge-neutral POPC/cholesterol liposomes (Lower) bind His-tagged MiD49∆TM poorly and do not promote Drp1 flotation. As depicted in the cartoon gradient at the right, protein-bound liposomes float to the top of the 0.5 M sucrose layer. (C) (Upper) In the presence of MiD49∆TM, Drp1 forms ordered polymers (arrows) with a diameter of 14.9 ± 1.5 nm. (Lower) Periodicity (∼5 nm) measured along the length of the Drp1:MiD49 polymers. (D) Effect of Drp1:MiD49 (molar:molar) ratios on polymer assembly. Decreasing MiD49 concentration reduces the formation of narrow (14.9-nm) polymers (white arrowheads) and increases the diameter of larger (34.4-nm) polymers (black arrowheads).
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