Structure-function analysis of Torsin ATPases in the context of the membrane

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 DESCRIPTION (provided by applicant): Torsins are essential members of the AAA+ (ATPases associated with a variety of cellular activities) superfamily and have been implicated in protein quality control, modulation of membrane morphology, and nuclear envelope dynamics. Four different Torsin proteins are known in humans. For years Torsins were thought to lack ATPase activity. Correspondingly, the precise cellular functions and the mechanistic roles of Torsin ATPases remained largely elusive. This gap in our knowledge hinders a comprehensive understanding of the etiology of congenital disorders caused by mutations in the Torsin system, including the severe movement disorder DYT1 dystonia. Importantly, our recent work has revealed that Torsins are ATPases whose activity requires LAP1 or LULL1, which are membrane-spanning cofactors that associate with Torsins to form a composite, membrane-spanning machine. Advances in our functional and mechanistic understanding of Torsin ATPases will require (i) definition of the structure and dynamics of the membrane-bound Torsin/cofactor assembly, (ii) identification of the cellular targets of the Torsin/cofactor machinery, and (iii) functional dissection of Torsin/cofactor action on substrates. In this proposal, we will capitalize on our established proteoliposome system to investigate the structural dynamics of full-length Torsin assembly with and without its cofactors via cryo-electron microscopy (Aim 1). We will utilize a novel methodology to overcome the poor solubility of Torsin substrates, allowing for substrate identification via a subtractive proteomic approach (Aim 2). The substrates, and their dependence on Torsin/cofactor action, will be analyzed in a cellular context, using genetic deletions in concert with different imaging techniques, as well as in reconstituted systems (Aim 3). The elucidation of Torsin function and mechanism -as well as its dysfunction resulting from disease-associated mutations- will enable the development of targeted therapies for the treatment of Torsin-related movement disorders, which are the most common inherited movement disorders known.