A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli

“…Human Drp1 (isoform 3) WT, ΔCT4, and ΔCT6 subcloned in pRSET C (Invitrogen), CT+ subcloned in pET21b (Novagen), and CT+* subcloned in pET Biotin His 6 FLASH (Addgene Plasmid #30184) were expressed and purified using a combination of His 6 -affinity and ion exchange chromatography as previously described 26 , 71 . 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) 72 and produced using the same protocol.…”

Section: Methodsmentioning

confidence: 99%

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

Pérez-Jover,

Rochon,

et al. 2024

Nat Commun

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Human Drp1 (isoform 3) WT, ΔCT4, and ΔCT6 subcloned in pRSET C (Invitrogen), CT+ subcloned in pET21b (Novagen), and CT+* subcloned in pET Biotin His 6 FLASH (Addgene Plasmid #30184) were expressed and purified using a combination of His 6 -affinity and ion exchange chromatography as previously described 22 , 54 . 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.…”

Section: Methodsmentioning

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

View full text Add to dashboard Cite

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.

show abstract

A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli

Cited by 4 publications

References 12 publications

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

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

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

Contact Info

Product

Resources

About