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GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

ABSTRACT: Membrane fusion of the ER is catalyzed when atlastin GTPases anchored in opposing membranes dimerize and undergo a crossed over conformational rearrangement that draws the bilayers together. Previous studies have suggested that GTP hydrolysis triggers crossover dimerization, thus directly driving fusion. In this study, we make the surprising observations that WT atlastin undergoes crossover dimerization before hydrolyzing GTP and that nucleotide hydrolysis and Pi release coincide more closely with dimer disassembly. These findings suggest that GTP binding, rather than its hydrolysis, triggers crossover dimerization for fusion. In support, a new hydrolysis-deficient atlastin variant undergoes rapid GTP-dependent crossover dimerization and catalyzes fusion at an initial rate similar to WT atlastin. However, the variant cannot sustain fusion activity over time, implying a defect in subunit recycling. We suggest that GTP binding induces an atlastin conformational change that favors crossover dimerization for fusion and that the input of energy from nucleotide hydrolysis promotes complex disassembly for subunit recycling.

SUBMITTER: Winsor J

PROVIDER: S-EPMC6279388 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

Winsor James J Machi Ursula U Han Qixiu Q Hackney David D DD Lee Tina H TH

The Journal of cell biology 20180924 12

Membrane fusion of the ER is catalyzed when atlastin GTPases anchored in opposing membranes dimerize and undergo a crossed over conformational rearrangement that draws the bilayers together. Previous studies have suggested that GTP hydrolysis triggers crossover dimerization, thus directly driving fusion. In this study, we make the surprising observations that WT atlastin undergoes crossover dimerization before hydrolyzing GTP and that nucleotide hydrolysis and Pi release coincide more closely wi ...[more]

PMID: 30249723

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Project description:The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.

Dataset Information

GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

Publications

GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

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