Affinity Purification and Functional Characterization of Dynamin-Related Protein 1

Clinton, Ryan W.; Bauer, Brianna L.; Mears, Jason A.

doi:10.1007/978-1-0716-0676-6_4

Cited by 12 publications

(10 citation statements)

References 14 publications

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“…Drp1 has been shown to exist as a mixture of dimers and tetramers in solution in a concentration-dependent manner, and the dimer state represents the core unit of the larger helical machinery 16 . For this study, WT Drp1 (isoform 1) was expressed in and isolated from E. coli using established methods 17 . This particular isoform is the second longest and includes the B insert (exons 16 and 17).…”

Section: Resultsmentioning

confidence: 99%

“…Drp1 Isoform 1 (UniProt ID O00429-1) was cloned into the pCal-N-EK vector as described previously 17 , 22 . Site-directed mutagenesis for Drp1 R456E and Y493A was completed to introduce each mutation into this construct individually using the QuikChange Lightning kit (Agilent) with primers from Integrated DNA Technologies (Coralville, IA).…”

Section: Methodsmentioning

confidence: 99%

“…The basal GTPase activity of Drp1 was measured using a colorimetric assay to detect released phosphate, as described previously 17 , 22 . Briefly, Drp1 (500 nM final) was diluted to 2.4X with Assembly Buffer (25 mM HEPES (KOH) pH 7.5, 150 mM KCl, 10 mM β-mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

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Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

Rochon,

Bauer,

Roethler

et al. 2024

Nat Commun

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Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

Rochon,

Bauer,

Roethler

et al. 2024

Nat Commun

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“…Previous studies examining the G363D variant have reported mixed hydrolysis results including no effect on hydrolytic activity ( Clinton et al, 2016 ), or impaired hydrolysis ( Tanaka et al, 2006 ; Chang et al, 2010 ). Given these discrepancies, it cannot be ruled out that differences in GTP hydrolysis may be due to variations in recombinant protein constructs or preparation methods (e.g., DRP1 isoforms, N- versus C-terminal tags, and calmodulin versus histidine purification tags) ( Clinton et al, 2020 ). One might anticipate that increased GTP hydrolysis would result in increased fission intracellularly.…”

Section: Discussionmentioning

confidence: 99%

NovelDNM1Lvariants impair mitochondrial dynamics through divergent mechanisms

et al. 2022

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Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene DNM1L, affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological DNM1L variants impair mitochondrial network maintenance by divergent mechanisms.

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“…Drp1 Isoform 3 (UniProt ID O00429-4) was cloned into the pCal-N-EK vector as described previously (Clinton et al, 2020; Clinton et al, 2016). Drp1-3 was expressed in BL21(DE3) Star Escherichia coli.…”

Section: Methodsmentioning

confidence: 99%

The Structure of the Drp1 Lattice on Membrane

Peng,

Rochon,

Stagg

et al. 2024

Preprint

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Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were hampered by heterogeneity, leaving a critical gap in the understanding of the mitochondrial fission mechanism. Here we present a cryo-electron microscopy structure of full length human Drp1 decorated on membrane tubules. Using the reconstruction of average subtracted tubular regions (RASTR) technique, we report that Drp1 forms a locally ordered lattice along the tubule without global helical symmetry. The filaments in the lattice are similar to dynamin rungs with conserved stalk interactions. Adjacent filaments are connected by GTPase domain interactions in a novel stacked conformation. Additionally, we observe contact between Drp1 and membrane that can be assigned to variable domain sequence. We identified two states of the Drp1 lattice representing conformational changes related to membrane curvature differences. Together these structures revealed a putative mechanism by which Drp1 constricts mitochondria membranes in a stepwise, "ratchet" manner.

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Affinity Purification and Functional Characterization of Dynamin-Related Protein 1

Cited by 12 publications

References 14 publications

Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

NovelDNM1Lvariants impair mitochondrial dynamics through divergent mechanisms

The Structure of the Drp1 Lattice on Membrane

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