Muller, Joyce Maja Miriam; (2001) Reverse genetic analysis of two membrane-fusion ATPases, NSF and p97. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the biochemical and molecular genetic characterisation of two related ATPases, NSF and p97. Both proteins belong to the AAA family, whose members function in a wide range of cellular processes including transcription, replication, cell division, protein folding and degradation. Despite the clear importance of many AAA proteins, details for cellular and biochemical functions of many family members, including NSF and p97, have remained elusive. The role of NSF in membrane fusion and trafficking in both the endocytic and exocytic pathways is undisputed, however the full extent and timing of its biochemical function is still unclear. A conceptual view of NSF is that it is a machine, which regulates the controlled assembly and disassembly of multimeric protein complexes. To relate the biochemical and functional aspects of NSF 1 introduced a mutation, based on a temperature-sensitive mutation of Drosophila NSF-1, termed comatose, into the mammalian NSF to study its function in a cell free assay for the fusion of Golgi membranes. By doing so, it was found that the mutation rendered the mammalian NSF temperature-sensitive for fusion of post-mitotic Golgi membranes and that an irreversible conformational change of the protein was responsible for its loss of function at the non-permissive temperature. Detailed investigations of the mammalian comatose protein using several biochemical assays uncovered the existence of a second novel NSF function that was necessary to drive post-mitotic Golgi membrane fusion, yet was distinct from NSF s proposed function as an ATPase-dependent SNARE disassembling machine. Furthermore, the two NSF-dependent steps required in membrane fusion were defined and characterised in more detail. Characterisation of the original Drosophila comatose protein revealed a biochemical and functional phenotype that was indistinguishable from the mammalian homologue, thus corroborating the authentic transplantation of the comatose phenotype into the mammalian NSF mutant, and the evolutionary conservation of the second function of NSF. Similar to NSF, the biochemical mode of action of the p97 ATPase is not thoroughly defined, yet unlike NSF, even the precise cellular context in which p97 functions is still unknown. To understand more fully the role played by p97 in mammals, I have characterised the structure and the expression pattern of the mouse p97 gene. Analysis of p97 protein levels in embryonic and adult mice revealed an unexpected degree of regulated expression and subcellular localisation in both proliferating and differentiated tissues suggesting a highly controlled and intermittent function for p97. A conditional gene targeting system was then established in murine ES cells and mice by using the Cre/LoxP recombination system to assess p97's physiological significance in vivo.

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