Reciprocal regulation between lunapark and atlastin facilitates ER three-way junction formation

Three-way junctions are characteristic structures of the tubular endoplasmic reticulum (ER) network. Junctions are formed through atlastin (ATL)-mediated membrane fusion and stabilized by lunapark (Lnp). However, how Lnp is preferentially enriched at three-way junctions remains elusiveHere, we showed that Lnp loses its junction localization when ATLs are deleted. Reintroduction of ATL1 R77A and ATL3, which have been shown to cluster at the junctions, but not wild-type ATL1, relocates Lnp to the junctions. Mutations in the Nmyristoylation site or hydrophobic residues in the coiled coil (CC1) of Lnp N-terminus (NT) cause mis-targeting of LnpConversely, deletion of the lunapark motif in the C-terminal zinc fin ger domain, which affects the homooligomerization of Lnp, does not alter its localizationPurified Lnp-NT attaches to the membrane in a myristoylation- dependent manner. The mutation of hydrophobic residues in CC1 does not affect membrane association, but compromises ATL interactionsIn addition, Lnp-NT inhibits ATL-mediated vesicle fusion in vitro. These results suggest that CC1 in Lnp-NT contacts junction-enriched ATLs for proper localization;subsequently, further ATL activity is limited by Lnp after the junction is formed. The proposed mechanism ensures coordinated actions of ATL and Lnp in generating and maintaining three-way junctions.