Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes - PubMed (original) (raw)
Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes
Xin Bian et al. Proc Natl Acad Sci U S A. 2011.
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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Structures of the cytosolic domain of human ATL1. (A) Scheme showing the domains of ATL1. 3HB, three-helix bundle; TMs, TM segments; CT, cytosolic tail. (B) Structure of the GDP-bound form of ATL1, corresponding to the postfusion state. The protomers in the dimer are shown in green and purple cartoon representation, superimposed on a space-filling model. The linkers (L-1 and L-2) between the GTPase domains and 3HBs (in pale colors) are highlighted. GDP is shown in orange stick representation, and magnesium ion is shown as a yellow sphere. The α-helices of the 3HBs are numbered. Right shows a different view of the dimer. (C) Structure of ATL1 crystallized with GDP and Pi, corresponding to the prefusion state. Lower shows a different view of the dimer.
Fig. 2.
Nucleotide-dependent dimerization of ATL1. (A) The size of wild-type (wt) cytATL1 (theoretical molecular mass 49.8 kDa) and of the R77E mutant (both at 0.05 mM) were determined by analytical ultracentrifugation in the presence of the indicated nucleotides. The estimated molecular masses are given above the peaks (in kDa). (B) Wild-type CytATL1 was subjected to gel filtration on a Superdex-200 column in the presence of the indicated nucleotides.
Fig. 3.
Nucleotide-dependent conformational changes of ATL1. (A) The two crystal structures were superimposed with their GTPase domains. The three-helix bundles (3HB) of the two ATL1 molecules are shown in gray and purple, respectively. Helices of the GTPase domain interacting with the 3HBs are shown in green. The bound nucleotide (in orange sticks) and the switch 2 region are indicated. Residue K345 is located in the α7-helix in the prefusion state and becomes accessible to trypsin in the postfusion state (arrowhead). (B) Stereoview of the prefusion interface between the GTPase domain and the 3HB. Interacting segments of the helices of the GTPase domain and of the α7-helix of the 3HB are shown in green, and relevant amino acids are indicated. The backbone of K345 is in red. The linker (L-1) is indicated. (C) As in B, but for the postfusion structure. (D) Confirmation of the conformational change by a trypsin-protection assay. Purified cytATL1 of wild-type (wt) or mutant human ATL1 was incubated with the indicated nucleotides (2 mM) and treated with protease. The samples in lanes 5–8 also contained 100 mM Pi. All samples were analyzed by SDS/PAGE and stained with Coomassie blue. The arrows indicate the full-length form and a protected fragment, respectively. (E) As in D, but with full-length Drosophila ATL, reconstituted into proteoliposomes. The vesicles were floated in a sucrose gradient before analysis by trypsin digestion. Pi was added at 500 mM. (F) Purified cytATL1 with a cysteine at position 357 of the 3HB was treated with the bifunctional cross-linker BMOE in the presence of the indicated nucleotides. Non-cross-linked protein (*) and cross-linked dimer (**) are indicated. (G) Purified cytATL1 with cysteines at positions 192 and 348 was incubated with the oxidant diamide in the presence of the indicated nucleotides. Where indicated, the disulfide bridge was reduced with β-mercaptoethanol (BME) before nonreducing SDS/PAGE.
Fig. 4.
Membrane fusion with wild-type (wt) and mutant ATL. (A) Full-length wt Drosophila ATL or mutants that affect dimer formation of human cytATL1 were reconstituted at equal concentrations into donor and acceptor vesicles. Reconstitution of the proteins was efficient as shown by flotation (
Fig. S10
) and resulted in all ATL molecules having their cytosolic domains exposed (Fig. 3_E_). GTP-dependent fusion of donor and acceptor vesicles was followed by the dequenching of a fluorescent lipid present in the donor vesicles. Control experiments were performed in the absence of Mg2+ or presence of GDP instead of GTP. (B) The fusion of vesicles containing full-length wt ATL was determined in the presence of increasing concentrations of the cytosolic domain (cytATL). The molar ratios are indicated. GST was used as a control. (C) As in A, but with mutants that affect GTPase activity or cause HSP (F126S, H222P, I290S). For comparison, the data in A for wt ATL are replotted. (D) As in C, but with mutants that affect the interaction between the GTPase domain and the three-helix bundle. ATL lacking the cytosolic tail (1–476) was also tested. (E) Mutations causing HSP were mapped into the postfusion structure (blue). The effect of mutation I315S (red) can only be explained by the prefusion structure. (F) The disease mutations were mapped into the prefusion structure.
Fig. 5.
A model for ATL-mediated homotypic membrane fusion. See Discussion for details. GTP and GDP molecules are indicated as magenta and yellow spheres, respectively. G, GTPase domain, 3HB, three-helix bundle, TMs, TM domains, L, linker.
References
- Baumann O, Walz B. Endoplasmic reticulum of animal cells and its organization into structural and functional domains. Int Rev Cytol. 2001;205:149–214. - PubMed
- Shibata Y, Voeltz GK, Rapoport TA. Rough sheets and smooth tubules. Cell. 2006;126:435–439. - PubMed
- Lee C, Chen LB. Dynamic behavior of endoplasmic reticulum in living cells. Cell. 1988;54:37–46. - PubMed
- Du Y, Ferro-Novick S, Novick P. Dynamics and inheritance of the endoplasmic reticulum. J Cell Sci. 2004;117:2871–2878. - PubMed
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