Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of 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 orthologue. 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.

For all assays, proteoliposomes were mixed with A100 buffer containing 5 mM MgCl₂ and then pre-incubated at 37°C for 5 minutes in a 96-well plate. Following pre-incubation, the plate was placed in a Spark plate reader (Tecan), two baseline readings were taken, and then 2 mM GTP or A100 buffer of equal volume was added with a multichannel pipette. For 60 minutes NBD dequenching was monitored at 10-second intervals (excitation at 460 nm, dequenching at 538 nm). Maximum possible dequenching was determined after 60 minutes via the addition of 0.5% Anapoe X-100, with two more cycles read on the plate reader after Anapoe addition. F_max was calculated by taking the average of these two post-Anapoe cycles. Fusion calculations were performed using the function: Fusion=((Fluorescence observed-Initial fluoresence observed)/Maximum fluorescence) x 100.

Microsoft Excel.