Mechanism and Role of Membrane Fusion by the Atlastin GTPase

Carnegie-Mellon University, Pittsburgh PA

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Abstract

Project Summary The endoplasmic reticulum (ER) is a dynamic membrane bound organelle whose polygonal branched network morphology is required for optimal protein targeting and secretion, lipid droplet formation and autophagy, among others. The branched structure of the ER is sustained through recurring membrane tubule extension events followed by homotypic tubule fusion events. The fusion events are catalyzed by the dynamin related ER membrane anchored GTPase atlastin (ATL). Absence of ATL activity in cells causes loss of ER network structure, while purified Drosophila ATL protein reconstituted into synthetic vesicles is sufficient to drive vesicle fusion. Vertebrates encode three distinct ATL paralogs. ATL2/3 are expressed broadly across diverse tissues while ATL1 is predominantly neuronal. In humans, mutations in ATL1 and ATL3 cause Hereditary Spastic Paraplegia (HSP) and Hereditary Sensory Neuropathy (HSN), respectively. Given the vital role of the ATL proteins in ER network homeostasis and their link to human disease, it is important to understand the respective roles of the paralogs and their regulation. However, while the study of Drosophila ATL has enabled a detailed understanding of the core ATL fusion mechanism, information regarding the human ATL1/2/3 paralogs, has been scant. This has been, in large part, due to a failure to reconstitute membrane fusion by any human paralog in vitro. In a recent breakthrough, our lab successfully reconstituted the fusion activity of all three human ATL paralogs. In the process, we discovered extensive autoinhibitory regulation of ATL1/2 by their variable C-terminal extensions. For ATL2, our preliminary results suggest that the inhibitory C-terminus forms a membrane inserted amphipathic helix that holds ATL2 in an orientation incompatible with fusion. For ATL1, C-terminal autoinhibition is less potent. However, our preliminary results also show that ATL1 both regulates, and is regulated by another HSP protein REEP1. ATL1 promotes membrane tubulation by REEP1, while REEP1 suppresses ATL1 fusion. In addition, a third HSP protein M1-spastin, a microtubule regulator, was reported to activate ATL1 fusion. These findings lead to a hypothesis that the three HSP proteins regulate each other, possibly to coordinate on another's ER structuring function. This proposal seeks to both unravel the autoinhibitory mechanisms controlling ATL1/2 fusion activity in diverse cell types (aim 1), and to test our hypothesis that ATL1, REEP1 and M1-spastin participate in a tripartite network to maintain ER homeostasis in neurons (aim 2).