Reply to Roy and Pucadyil: A gain of function by a GTPase-impaired Drp1

Pérez-Jover, Isabel; Mohan, Pooja Madan; Ramachandran, Rajesh; Shnyrova, Anna V.

doi:10.1073/pnas.2202391119

Cited by 2 publications

(6 citation statements)

References 11 publications

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“…As previously shown 17 , 25 , WT Drp1 did not effectively catalyze NT fission on its own (Fig. 4a, b ).…”

Section: Resultssupporting

confidence: 81%

“…These non-native extensions were introduced to isolate the backbone carboxylate moiety of the C-terminal residue, a requirement for high-affinity PDZ domain binding 22 , from the predicted PDZ domain recognition sequence in Drp1 ( 696 THLW 699 ) 18 . In addition, we sought to determine the influence of non-native C-terminal extensions on Drp1 structure and function, which in past studies have produced confounding and conflicting results 23 – 25 . To enable purification, WT Drp1 and select CT variants were modified at the N-terminus with a His 6 affinity tag (see Methods), which as previously shown 26 , 27 did not affect Drp1 self-assembly or GTPase activity in vitro.…”

Section: Resultsmentioning

confidence: 99%

“…2d ). Trp emission is highly sensitive to its microenvironment and therefore serves as an accurate probe of protein conformation or conformational changes 25 , 26 . When excited selectively at λ = 295 nm, the Trp emission spectrum is blue-shifted (peaking at shorter wavelengths) when present in a nonpolar environment, and red-shifted (peaking at longer wavelengths) when exposed to a polar or aqueous milieu.…”

Section: Resultsmentioning

confidence: 99%

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Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif

Pérez-Jover,

Rochon,

et al. 2024

Nat Commun

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The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.

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“…As previously shown 17 , 25 , WT Drp1 did not effectively catalyze NT fission on its own (Fig. 4a, b ).…”

Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif

Pérez-Jover,

Rochon,

et al. 2024

Nat Commun

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“…As previously shown 17,27 , WT Drp1 did not mediate NT fission on its own, but instead effectively constricted NTs to a final radius of 14 ± 2 nm independent of starting (initial) NT radii (Fig. 4A, Supplementary Movie 1).…”

Section: Ct-slim Controls Drp1-catalyzed Membrane Fission In Vitrosupporting

confidence: 77%

“…Mouse CT+ identical to human CT+ in length (699 aa residues) and also in composition except for eight alternative residues within the G domain (3) and VD (5) was obtained from Addgene (Plasmid # 72927) 55 and produced using the same protocol. The CT+ variant was referred to as Drp1-C in our previous study 27 . CT+ sh was produced from mouse CT+ by human rhinovirus (HRV) 3C protease cleavage.…”

Section: Protein Productionmentioning

confidence: 99%

Allosteric control of dynamin-related protein 1-catalyzed mitochondrial fission through a conserved disordered C-terminal Short Linear Motif

Pérez-Jover¹,

Rochon²,

Hu³

et al. 2023

Preprint

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The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial fission, but the regulatory mechanisms remain ambiguous. Here we found that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, leading to the fission of model membranes in vitro. In vivo, the availability of the native CT-SLiM is a requirement for productive mitochondrial fission, as both non-native extension and deletion of the CT-SLiM severely impair its progression. Thus, contrary to prevailing models, Drp1-catalyzed mitochondrial fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.

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Reply to Roy and Pucadyil: A gain of function by a GTPase-impaired Drp1

Cited by 2 publications

References 11 publications

Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif

Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif

Allosteric control of dynamin-related protein 1-catalyzed mitochondrial fission through a conserved disordered C-terminal Short Linear Motif

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