INTRINSIC AND EXTRINSIC REGULATORY MECHANISMS OF THE DYNAMIN-RELATED G PROTEIN ATLASTIN: IMPLICATIONS IN CELLULAR FUNCTION AND HUMAN HEALTH
Kelly, Carolyn McCartney
The complex and diverse biochemical processes that support life require both the physical separation and dynamic communication that membranes provide. Critical membrane dynamics are achieved through fusion and fission events that are regulated and catalyzed by various classes of proteins, including dynamins. Here we present studies of the dynamin-related GTPase, atlastin (ATL), which catalyzes homotypic fusion of peripheral ER tubules to form and maintain its distinctive reticular structure. Humans have three isoforms (ATL1-3), all localizing to and functioning in the ER. Mutations in atl1 and atl3 are causative in two hereditary neuropathies, hereditary spastic paraplegia (HSP) and hereditary sensory neuropathy (HSN), caused by length-dependent axonal degeneration. Previous structural and biochemical studies have converged on a mechanistic model of how ATLs undergo GTP binding- and hydrolysis-dependent conformational changes coupled with dimerization in a manner that supports membrane fusion. However, questions remain regarding how this model translates to ATL’s mechanism in a complex biological environment, including what mechanisms dictate how ATL favors GTP hydrolysis-dependent dimerization across trans membranes rather than cis membrane dimerization, which is a futile cycle in regards to membrane fusion, and the modes of ATL regulation that result in re-structuring of ER morphology in response to various cell signals, especially cell division. Here, we present two novel modes of ATL1 regulation, both intrinsically through an uncharacterized N-terminal hypervariable region (HVR) and extrinsically through phosphorylation of several conserved serine resides within this motif. Our evidence reveals that the structured ATL1 HVR provides a novel intermolecular contact that supports innate aspects of its membrane tethering activity. We also establish that ATL1 HVR-dependent phosphorylation has effects on both its kinetic behavior and cellular function, and using a kinase screen, have identified a list of candidate ATL1 modifiers. We have also structurally and biochemically characterized a novel HSP-associated ATL1 mutation, resulting in a whole codon insertion. We have uncovered a gain-of-function pathogenic mechanism yielding increased membrane tethering activity and altered ER sub-localization, while GTP hydrolysis remains unaffected. We propose the causative mechanism lies in the disruption of a pre-hydrolysis conformational state, leaving the mutant protein primed for faster nucleotide-dependent dimerization. These studies expand our current understanding of ATL’s functional mechanisms in the cell and our appreciation of how this translates to human neuronal health.
Brown, William J.; Sevier, Carolyn S.
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis