Structural and functional studies of membrane remodeling machines

Kalia, Raghav; Talledge, Nathaniel; Frost, Adam

doi:10.1016/bs.mcb.2015.02.007

Cited by 7 publications

(7 citation statements)

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“…Protein purification was performed as described 52 . Briefly, plasmids containing the WT DRP1 or MID49 126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli .…”

Section: Methodsmentioning

confidence: 99%

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Structural basis of mitochondrial receptor binding and constriction by DRP1

Kalia

Wang

Ali

et al. 2018

Nature

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Summary Mitochondrial inheritance, genome maintenance, and metabolic adaptation depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryoEM structure of human, full-length DRP1 coassembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes polymerization of a linear DRP1 filament with MID49/MID51. Following coassembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.

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“…Protein purification was performed as described 52 . Briefly, plasmids containing the WT DRP1 or MID49 126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli .…”

Section: Methodsmentioning

confidence: 99%

“…Liposomes were made as described before 52 . Briefly, 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of mitochondrial receptor binding and constriction by DRP1

Kalia

Wang

Ali

et al. 2018

Nature

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272

273

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“…Human dynamin1 was purified as described from baculovirus-infected Sf9 cells expressing recombinant dynamin1, via affinity purification with the SH3 domain of rat amphiphysin1 ( Kalia et al, 2015 ; Stowell et al, 1999 ).…”

Section: Methodsmentioning

confidence: 99%

Structural inhibition of dynamin-mediated membrane fission by endophilin

Hohendahl

Talledge

Galli

et al. 2017

eLife

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Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here, we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission.

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“…SF9 cells were infected with viruses and then grown for 3 days at 27°C, and then centrifuged at 2,000 g for 5 minutes. DNM2 recombinant proteins were purified with amphiphysin-SH3 bound to Glutathione-Sepharose 4B beads (GE Healthcare) as previously described (46). BIN1 recombinant protein was produced from pGEX6P1 plasmid in E. coli BL21.…”

Section: Animal Experimentsmentioning

confidence: 99%