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. 2015 Feb;6(2):139-46.
doi: 10.1007/s13238-014-0118-0. Epub 2014 Nov 20.

Comparison of Human and Drosophila Atlastin GTPases

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Free PMC article

Comparison of Human and Drosophila Atlastin GTPases

Fuyun Wu et al. Protein Cell. .
Free PMC article

Abstract

Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.

Figures

Figure 1
Figure 1
Crystal structure of the cytosolic domain of Drosophila ATL. (A) Schematic diagrams of Drosophila ATL and human ATL1. (B) Human ATL1 dimers observed in crystal form 1 (PDB code: 3QNU, left, GDP-bound), form 2 (PDB code: 3QOF, middle, GDP-bound in the presence of inorganic phosphate), and form 3 (PDB code: 4IDO, right, GDP/AlF4-bound or GppNp-bound) are shown. The protomers in the dimer are shown in green and purple cartoon representation. (C) Structure of the GDP-bound form of Drosophila ATL. The protomers in the dimer are shown in green and purple cartoon representation. GDP is shown in orange stick representation, and magnesium ion is shown as a yellow sphere. 3HB, three-helix bundle. (D) Structural comparison of human and Drosophila ATLs. Human ATL1 (hsATL1) is shown in pink and Drosophila ATL (dmATL) is shown in green. GDP in dmATL is shown in orange, and magnesium ion is shown in yellow. (E) As in C, but with a dimer. Superposition was performed using the protomers on the left
Figure 2
Figure 2
Comparison of the active site. Stereoview of the superimposed active sites of Drosophila ATL (colored as in Fig. 1C) and human ATL1 (in pink, PDB ID code 3QOF). Critical residues are highlighted. The nucleotides are shown in stick representation and the magnesium ions as spheres
Figure 3
Figure 3
Functional tests of Drosophila ATL in mammalian cells. (A) COS-7 cells were transfected with siRNA oligonucleotides as indicated. Levels of endogenous ATL2 and ATL3 or exogenously expressed Drosophila ATL and human ATL1 were determined by immunoblotting. GAPDH was used as a loading control. Asterisk (*) indicates a non-specific band. (B) The ER morphology of COS-7 cells was visualized using calreticulin, an endogenous luminal ER protein, and indirect immunofluorescence using a confocal microscope. Scale bar = 10 μm. (C) As in (B), but with double-depleted COS-7 cells transfected with Flag-human ATL1, or Drosophila ATL. (D) The ER morphology of samples shown in (B) and (C) was categorized as “normal” or “unbranched”. A total of 80–150 cells were counted for each sample. All graphs are representative of three repetitions
Figure 4
Figure 4
Biochemical comparison of cytATLs. (A) Nucleotide-dependent dimerization of the N-terminal cytosolic domain of hsATL1 was determined at a concentration of 0.03 mmol/L by analytical ultracentrifugation in the presence of the indicated nucleotides. (B) As in (A), but with dmATL. (C) The GTPase activities of 1 μmol/L, 2 μmol/L, and 5 μmol/L of N-terminal cytosolic domains of human ATL1 (hsATL1) and Drosophila ATL (dmATL) were measured by phosphate release at saturating concentrations of GTP (0.5 mmol/L). All graphs are representative of three repetitions
Figure 5
Figure 5
Comparison of membrane fusion in vitro. (A) Full-length ATLs were reconstituted into proteoliposomes. Flotation in a sucrose gradient indicated efficient reconstitution of the proteins. (B) Full-length ATLs were reconstituted at equal concentrations into donor and acceptor vesicles. The lipid to protein ratio used for each protein is indicated. Fusion was monitored by dequenching the NBD-labeled lipids present in the donor vesicles and was initiated by the addition of GTP. Control experiments were performed using chimeric ATL in the absence of Mg2+ or the presence of GDP or GppNp instead of GTP. (C) The GTPase activities of purified full-length ATLs (1 μmol/L, 2 μmol/L, and 5 μmol/L) were measured by phosphate release at saturating concentrations of GTP (0.5 mmol/L). All graphs are representative of three repetitions

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