Regulation of human atlastin by the c-terminus

The endoplasmic reticulum (ER) is a continuous membrane network that spans the entirety of the cell. It consists of a balance of sheets and tubules. Both tubules and sheets are constantly remodeled by movement along microtubules and their subsequent fusion. Because the atlastin GTPases are required for ER structure and purified Drosophila atlastin is sufficient for fusion in vitro, ER fusion in mammalian cells has been assumed to be mediated by atlastin. This has important implications for understanding why mutations in human atlastins cause disease. However, the lack of in vitro fusion activity of the human atlastins raised the question of whether it is fusogen and impeded understanding of the basis of diseases linked to mutations in atlastin. Importantly, all previous studies involved purifying the human atlastins from its non-native environment in E. coli. I was successfully able to purify human atlastins from a mammalian cell line and assay for fusion activity. I showed that human atlastin1 (ATL1) purified from mammalian cells is indeed sufficient for fusion. ATL1 exhibited fusion activity and disease variants showed alterations in fusion. This result lays the foundation for further studies of human atlastins and their disease variants in the context of the membrane. Surprisingly, the more ubiquitously expressed ATL2 paralog had negligible fusion activity. This was unexpected because the GTPase domains of ATL1 and ATL2 are 75% identical, the ATL2 soluble domain dimerizes and hydrolyses GTP at rates comparable to ATL1, and most cell types rely on ATL2/3 rather than ATL1. Sequence analysis revealed a non-conserved C-terminal extension on ATL2, and deletion of this extension restored maximal fusion activity suggesting a novel mode of regulation by the C-terminus. Charge reversal of residues in this inhibitory domain of ATL2 strongly activated its fusion activity and overexpression of this disinhibited version caused ER collapse. Finally, neurons express an alternate ATL2 splice isoform whose sequence differs in this inhibitory domain, and this form showed full fusion activity. These published findings (chapter two) reveal autoinhibition and alternate splicing as regulators of atlastin mediated ER fusion. Additional unpublished work herein investigated the role of crossover in fusion. Utilizing a FRET based approach, I sought to determine if crossover drives fusion by visualizing crossover in the context of the full-length protein in membranes. My results suggest that crossover formation is not sufficient to catalyze fusion. These data and discussion of their caveats are presented in chapter three.