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. 2015 Sep 14;210(6):961-72.
doi: 10.1083/jcb.201502078.

Structures of the Yeast Dynamin-Like GTPase Sey1p Provide Insight Into Homotypic ER Fusion

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Free PMC article

Structures of the Yeast Dynamin-Like GTPase Sey1p Provide Insight Into Homotypic ER Fusion

Liming Yan et al. J Cell Biol. .
Free PMC article

Abstract

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4(-) but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.

Figures

Figure 1.
Figure 1.
Structures of the cytosolic domain of Sey1p. (A) Scheme showing the domains of Sey1p from C. albicans (caSey1p) and S. cerevisiae (scSey1p). Residue numbers for each domain are indicated. The domains of caSey1p are colored, respectively. 3HB, three-helix bundle; TMs, transmembrane segments; CT, cytosolic tail. (B) Structure of the GDP/AlF4-bound form of caSey1p. The protomers in the dimer are shown in colored (as in A) and gray cartoon representation. The linker between the GTPase and stalk domain and the long loop in the stalk domain are highlighted. GDP is shown in pink stick representation, AlF4 is in cyan sticks, and magnesium ion is shown as a lime sphere. The 3HBs in the stalk domain are numbered, and the secondary structure elements are labeled. (C) As in B, but for structure of caSey1p crystallized with GDP. (D) A topology plot of the stalk domain, colored as in A.
Figure 2.
Figure 2.
Nucleotide-dependent dimerization of Sey1p. (A) The interface between the two GTPase domains. The cartoon representation is colored as in Fig. 1. Key residues are indicated as sticks. The components of the second protomer (gray) are labeled with a prime symbol. (B) As in A, but for the interface between the two stalk domains. (C) The size of cyt-scSey1p (40 µM, theoretical molecular mass 80.8 kD) or cyt-hsATL1 (40 µM, theoretical molecular mass 51.7 kD) was determined by AUC in the presence of the indicated nucleotides. The estimated molecular masses are given above the peaks in kilodaltons. The data shown are from a single representative experiment out of three repeats. (D) Full-length wild-type (wt) scSey1p was reconstituted at equal concentrations into donor and acceptor vesicles (protein/lipid molar ratio of 1:200). Fusion of donor and acceptor vesicles was followed by the dequenching of an NBD-labeled lipid present in the donor vesicles. Nucleotides, if indicated, were added at time 0. The data shown are from a single representative experiment out of four repeats. (E) As in D, but with stalk mutants. Δ3/4 stalk, deletion of residues 378–675 in scSey1p; Δ1/2 stalk, deletion of 462–675; ΔLoop, deletion of 613–636. The data shown are from a single representative experiment out of three repeats. (F) scSey1p-containing proteoliposomes, which also contained either Rhodamine- or Oregon green–labeled lipids, were mixed at a 1:1 ratio (total lipid concentration: 0.6 mM). One aliquot was analyzed immediately, a second was taken after incubation at 37°C with 1 mM GTP for 5 min, and a third was obtained after incubation with GTP followed by addition of 10 mM EDTA. All samples were diluted, spotted onto a coverslip, and visualized by confocal microscopy. Bar, 5 µm. (G) As in F, but with 1 mM GMP-PNP. (H) As in F, but with GDP/AlF4. Arrows indicate tethered/fused vesicles.
Figure 3.
Figure 3.
Nucleotide-dependent conformational changes of Sey1p. (A) Cartoon representation of the active site. The GDP/AlF4 state is colored in red and the GDP state in gold. The four signature motifs are highlighted. Note that switch 1 and switch 2 in the GDP state are partly disordered. The pairing protomer in the GDP/AlF4 state is shown in gray. (B) Superposition of the Sey1p structures in the GDP/AlF4 state (red) and the GDP state (gold). The movement of the GTPase domain is highlighted. (C) The GTPase-linker interface in the GDP/AlF4 state. The cartoon representation is colored as in Fig. 1. Key residues are shown in sticks. (D) As in C, but for the GTPase–stalk interface in the GDP state. (E) Purified cytosolic domains of scSey1p with cysteines at positions 297 and 298 or at positions 274 and 298 were treated with the oxidant diamide in the presence of the indicated nucleotides. Non–cross-linked protein (single asterisks) and cross-linked dimer (double asterisks) are indicated. Where indicated, the disulfide bridge was reduced with β-mercaptoethanol (BME) before nonreducing SDS/PAGE. (F) As in E, but with wild type (wt) or mutant (cysteines at positions 407 and 467) of the cytosolic domain of caSey1p. (G) Fusion assays of the M274A mutant of scSey1p. Wild-type samples were included for comparison. (H) As in G, but with the mutants of L297A and V298A. The data shown in G and H represents independent experiments with three independently purified batches of protein.
Figure 4.
Figure 4.
Functional tests of Sey1p in cells. (A) COS-7 cells were transfected with siRNA oligonucleotides against hsATL2 and 3. The levels of these proteins were determined by immunoblotting. GAPDH was used as a loading control. IB, immunoblot. (B) The ER morphology of COS-7 cells was visualized using calreticulin, an endogenous luminal ER protein, and indirect immunofluorescence using a confocal microscope. (C) Myc-hsATL1, Myc-scSey1p, or Myc-caSey1p was expressed in COS-7 cells and its localization determined by anti-Myc antibodies (green) and compared with that of calreticulin (red) using indirect immunofluorescence and confocal microscopy. (D) The ER morphology of indicated samples was categorized as “normal” (as in the top image of B) or “unbranched” (as in the bottom image of B). A total of 80–150 cells were counted for each sample. All graphs are representative of at least three repetitions. wt, wild type. Bars, 10 µm.
Figure 5.
Figure 5.
Sey1p punctae formation. (A) Myc-scSey1p was expressed in COS-7 cells and its localization determined by anti-Myc antibodies and compared with that of calreticulin using indirect immunofluorescence and confocal microscopy. The right image shows an enlargement of the boxed region centered on the tubular ER network. Bars, 10 µm. (B) As in A, but with cells expressing Myc-scSey1p L233A. (C) As in A, but with cells expressing Myc-hsATL1. (D) As in A, but with cells expressing Myc-hsATL3. (E) As in A, but with cells expressing Myc-hsATL1 R77A. The arrowheads indicate the puncta localized at the three-way junctions of the ER network.
Figure 6.
Figure 6.
Comparison of homotypic membrane fusion mediated by Sey1p and ATL. (A) A model of Sey1p-mediated fusion. See Discussion for details. GTP and GDP molecules are indicated as cyan and blue spheres, respectively. (B) As in A, but for ATL. TMs, transmembrane segments; CT, cytosolic tail.

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