Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

Crosby, Daniel; Mikolaj, Melissa R.; Nyenhuis, Sarah B.; Bryce, Samantha; Hinshaw, Jenny E.; Lee, Tina H.

doi:10.1083/jcb.202107070

Cited by 13 publications

(47 citation statements)

References 56 publications

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“…To assay membrane fusion, we utilized D ATL protein purified from a HEK293-derived mammalian suspension cell line since our recent work revealed D ATL from these cells to be more active than protein purified from Escherichia coli ( Crosby et al. , 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis

Crosby

Lee

2022

MBoC

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Atlastin (ATL) GTPases undergo trans dimerization and a power stroke-like crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. We recently used the truncated cytoplasmic domain of wild-type Drosophila ATL ( DATL) and a novel hydrolysis deficient D127N variant in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. Here, we report that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. Our findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling.

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Section: Resultsmentioning

confidence: 99%

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis

Crosby

Lee

2022

MBoC

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“…For Drp1, target receptors and lipids likely fulfill these roles. Recent studies show that the extreme C-termini of the distantly related atlastins 49,50 , involved in ER membrane fusion, function in a similar autoregulatory capacity 50,51 . Thus, from an evolutionary standpoint, the extreme C-terminus may represent a critical, conserved, regulatory feature of all dynamin superfamily proteins (DSPs) 49 .…”

Section: Discussionmentioning

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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Section: Introductionmentioning

confidence: 99%

“…Despite substantial advances using D ATL, reconstitution of fusion activity by the human ATLs had proved refractory until recently, when our lab demonstrated that ATL1/2 expressed and purified from HEK cells, rather than E. coli , has robust fusion activity ( Crosby et al, 2022 ). Unexpectedly, fusion reconstitution also revealed that each contains a C-terminal autoinhibitory domain located just downstream of the required AH ( Crosby et al, 2022 ). For ATL2, the presence of the C-terminal extension inhibits fusion to the extent that the full-length purified protein lacks nearly all detectable activity, and removal of the extension increases the initial fusion rate by 500-fold.…”

Section: Introductionmentioning

confidence: 99%

Human atlastin-3 is a constitutive ER membrane fusion catalyst

Bryce

Stolzer

Crosby

et al. 2023

Journal of Cell Biology

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Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of the three human ATL paralogs (ATL1/2) are C-terminally autoinhibited implied that relief of autoinhibition would be integral to the ATL fusion mechanism. An alternative hypothesis is that the third paralog ATL3 promotes constitutive ER fusion with relief of ATL1/2 autoinhibition used conditionally. However, published studies suggest ATL3 is a weak fusogen at best. Contrary to expectations, we demonstrate here that purified human ATL3 catalyzes efficient membrane fusion in vitro and is sufficient to sustain the ER network in triple knockout cells. Strikingly, ATL3 lacks any detectable C-terminal autoinhibition, like the invertebrate Drosophila ATL ortholog. Phylogenetic analysis of ATL C-termini indicates that C-terminal autoinhibition is a recent evolutionary innovation. We suggest that ATL3 is a constitutive ER fusion catalyst and that ATL1/2 autoinhibition likely evolved in vertebrates as a means of upregulating ER fusion activity on demand.

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Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

Cited by 13 publications

References 56 publications

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis

Membrane fusion by Drosophila atlastin does not require GTP hydrolysis

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

Human atlastin-3 is a constitutive ER membrane fusion catalyst

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