Background:Atlastin is large GTPase that catalyzes the homotypic fusion of ER membranes. Results: In vitro and in vivo studies reveal that the C-terminal tail of Atlastin affects its function. Conclusion:The amphipathic C-terminal tail of Atlastin destabilizes lipid bilayers to promote membrane fusion. Significance: Describing the mechanism of Atlastin-mediated fusion is a critical step in our understanding of ER structure formation.

The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro. Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro. We also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.A ll members of the Orthomyxoviridae family, including the influenza viruses A-D, thogotovirus, and isavirus, encode a matrix protein called M1. In the influenza A virus, M1 is produced by a colinear transcript made from the gene segment 7 (1). As one of the most abundantly made viral proteins, the influenza A virus M1 plays multiple roles during the virus life cycle. Upon viral entry, the acidification of the viral interior in the endosome weakens the interaction between M1 and the viral ribonucleoprotein complexes (vRNPs), thus allowing M1-free vRNPs to be imported to the nucleus for viral RNA replication and transcription (2, 3). As infection proceeds, newly synthesized M1 enters the nucleus to mediate the nuclear export of nascent vRNPs. A "daisy-chain" complex is formed with the vRNP binding to the C-terminal domain of M1 (4) and the N-terminal domain of M1 interacting with the nuclear export protein (NEP), also called NS2. Through its nuclear export signal (NES), NEP is specifically recognized by the cellular exportin Crm1, which then facilitates the transport of vRNPs across the nuclear membrane to the cytoplasm in a RanGTP-dependent manner (5). It has been shown that M1 binding to vRNP in the nucleus is able to block mRNA transcription (4, 6). In the cytosol, M1 interacts with the cytoplasmic tails of the glycoproteins HA and NA. Such interactions promote M1 association with lipid raft membranes and...

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel a helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.

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