Membrane fusion is essential for a wide variety of biological processes. Studies on viral and SNARE fusion protein catalysts have revealed a common strategy by which proteins anchored in opposing membranes undergo favorable protein-folding reactions that draw the membranes into close apposition and drive the lipid rearrangements necessary for fusion. More recently, a new fusion paradigm has arisen with discovery that atlastin (ATL) a membrane-anchored dynamin-related GTPase can trigger fusion of synthetic liposomes, and is required for the branched morphology of the ER. ATL is distinct from previously studied fusion catalysts because it is a mechanochemical enzyme that couples hydrolysis of GTP to fusion catalysis. Importantly, while substantial progress has been made, basic questions remain unresolved and there is still little consensus on mechanism. In the presence of GTP, the N-terminal cytosolic domain of ATL undergoes trans dimerization and a crossover conformational change hypothesized to draw membranes sufficiently close together to drive fusion. However, no fusion is observed in the absence of an amphipathic helix within the C-terminal cytosolic tail of ATL, suggesting a sequential model in which crossover formation constitutes an upstream step for membrane docking, and the tail functions subsequently to drive lipid mixing. On the other hand, our recent work suggests that crossover dimerization provides the energy for fusion, but does not explain the role of the tail. Thus whether crossover serves primarily to mediate docking, or whether it drives fusion, needs to be resolved. Similarly, how GTP hydrolysis energizes the fusion reaction cycle is under debate. Prevailing models have held that the hydrolysis of GTP powers formation of the ATL crossover dimer directly for fusion. However, our recent work suggests that GTP hydrolysis serves to disassemble, rather than to assemble, the crossover dimer, and more likely serves to recycle the fusion machinery after the completion of fusion. This change constitutes a paradigm shift, and needs to be firmly established.
In aim 1 we will ascertain the role of crossover dimerization in fusion using FRET probes to monitor the timing of crossover dimerization relative to lipid mixing and determine whether crossover formation invariably coincides with fusion, and whether crossover formation requires the ATL tail.
In aim 2, we will extend our analysis of the GTP hydrolysis reaction cycle from the soluble phase to the context of membranes to ascertain whether the hydrolysis of GTP, as suggested by our new model, functions only after the completion of fusion for the purpose of subunit recycling. Altogether, the proposed studies promise to reveal broad mechanistic insights into how GTP-dependent fusion proteins catalyze membrane fusion as well as to uncover shared principles among disparate fusion catalysts. Also, because mutations in human ATL1 cause the motor neurological disorder HSP whose basis is not understood, these studies have the potential to shed light on disease causality and possibly also impact its therapeutics.
The proposed studies seek to use biochemical, biophysical and cell-based approaches to better understand the molecular mechanism by which the atlastin GTPase harnesses nucleotide hydrolysis to catalyze the fundamental process of ER membrane fusion. Mutations in atlastin1 cause the human motor neurological disorder hereditary spastic paraplegia (HSP). Therefore the outcomes will not only to provide insights into the principles by which a mechanochemical enzyme catalyzes membrane fusion, but will also reveal new information on the basis for HSP.
| Winsor, James; Machi, Ursula; Han, Qixiu et al. (2018) GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion. J Cell Biol 217:4184-4198 |
| Winsor, James; Hackney, David D; Lee, Tina H (2017) The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion. J Cell Biol 216:1321-1335 |
| Ulengin, Idil; Park, John J; Lee, Tina H (2015) ER network formation and membrane fusion by atlastin1/SPG3A disease variants. Mol Biol Cell 26:1616-28 |
| Faust, Joseph E; Desai, Tanvi; Verma, Avani et al. (2015) The Atlastin C-terminal tail is an amphipathic helix that perturbs the bilayer structure during endoplasmic reticulum homotypic fusion. J Biol Chem 290:4772-83 |
| Saini, Simran G; Liu, Chuang; Zhang, Peijun et al. (2014) Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration. Mol Biol Cell 25:3942-53 |
