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Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphosphatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a postfusion state. How the prefusion conformer transitions to the postfusion conformer is unknown. In this paper, we identify an intramolecular salt bridge mediated by two residues outside the GTPase domain near the point of rotation that converts the prefusion dimer to the postfusion state. Charge reversal of either residue blocked ER network branching, whereas a compensatory charge reversal to reestablish electrostatic attraction restored function. In vitro assays using the soluble domain revealed that the salt bridge was dispensable for GTP binding and hydrolysis but was required for forming the postfusion dimer. Unexpectedly, the postfusion conformation of the soluble domain was achieved when bound to the nonhydrolyzable GTP analogue guanosine 5'-[β,γ-imido]triphosphate, suggesting that nucleotide hydrolysis might not be required for the prefusion to postfusion conformational change.

Original publication

DOI

10.1083/jcb.201105006

Type

Journal article

Journal

J Cell Biol

Publication Date

14/11/2011

Volume

195

Pages

605 - 615

Keywords

Endoplasmic Reticulum, GTP Phosphohydrolases, Guanosine Triphosphate, HeLa Cells, Humans, Membrane Fusion, Models, Molecular, Protein Conformation, Salts, Solubility