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Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the crossover configuration.
The membrane-anchored atlastin GTPase couples nucleotide hydrolysis to the catalysis of homotypic membrane fusion to form a branched ER network. Trans dimerization between atlastins anchored in opposing membranes, accompanied by a crossover conformational change, is thought to draw the membranes together for fusion. Previous studies on the conformational coupling of atlastin to its GTP hydrolysis cycle have been carried out largely on atlastins lacking a membrane anchor. Consequently, whether fusion involves a discrete tethering step, and if so, the potential role of GTP hydrolysis and crossover in tethering remain unknown. Here, we used membrane-anchored atlastins in assays that separate tethering from fusion to dissect the requirements for each. We found that tethering depended on GTP hydrolysis; but, unlike fusion, it did not depend on crossover. Thus, GTP hydrolysis initiates stable head domain contact in trans to tether opposing membranes, whereas crossover formation plays a more pivotal role in powering the lipid rearrangements for fusion.