Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes

Abstract

The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.protein structure | membrane remodeling | organelle shaping | spastic paraplegia gene 3A | endoplasmic reticulum network formation

Cis and trans interactions between atlastin molecules during membrane fusion

“…10, 11). They contain cytosolic N-terminal GTPase (G) and helical bundle domains, followed by two closely spaced transmembrane (TM) segments and a cytosolic C-terminal tail (12)(13)(14). A role for the GTPases in ER fusion is suggested by the observation that their depletion or mutational inactivation leads to long, nonbranched tubules or fragmented ER (8, 9).…”

“…Crystal structures of ATL and biochemical experiments have not resulted in a coherent model. Initial structures of the N-terminal cytosolic domain of human atlastin-1 (ATL1) revealed two different conformations (13,14). In both conformations, a dimer is formed between the two G domains, but in one conformation (crystal form 2; SI Appendix, Fig.…”

“…In addition, the fusion of ER vesicles in Xenopus laevis egg extracts is prevented by ATL antibodies or a cytosolic fragment of ATL (8,15), and the fusion of the ER in mating yeast cells is delayed on SEY1 deletion (16). Most convincingly, proteoliposomes containing purified ATL, Sey1p, or RHD3 undergo GTP-dependent fusion in vitro (9,13,16,17). Defects in ATL-mediated ER fusion can cause hereditary spastic paraplegia, a neurodegenerative disease characterized by the shortening of the axons in corticospinal motor neurons, leading to progressive spasticity and weakness of the lower limbs (13,18).…”

“…S1), they are parallel to one another. These structures were interpreted as "prefusion" and "postfusion" states, in which the two ATL molecules would either tether two opposing membranes or sit in the same membrane after fusion (13).…”

“…Because the prefusion structure was crystallized in the presence of GDP and inorganic phosphate (P i ) and the postfusion structure was crystallized in the presence of GDP, it was proposed that GTP hydrolysis would induce this conformational change, resulting in the apposed membranes being pulled together for fusion (13). Both structures contained only bound GDP, however, raising doubts that the conformational change is induced by GTP hydrolysis.…”