Human Atlastin-3 is a Constitutive ER Membrane Fusion Catalyst

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.