Niewiarowski, A.; (2009) Subunit composition, assembly and regulation of human TIP48 and TIP49 complexes. Doctoral thesis , UCL (University College London).

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Abstract

The two closely related eukaryotic AAA+ proteins, TIP48 and TIP49, are essential components of large multi-protein complexes that are involved in diverse cellular processes. Specific mechanisms for their activity in transcriptional regulation, chromatin remodelling, DNA repair and apoptosis have not yet been characterised; however, different oligomeric forms, sub-cellular localisation and post translational modification of TIP48 and TIP49 may be important in co-ordinating these functions. The assembly of the human TIP48 and TIP49 into different oligomers was studied. Both proteins were mainly monomeric in the absence of nucleotide cofactors. Incubation with ATP or ADP in the presence of Mg2+ modified the distribution of TIP48 oligomers to favour hexamers, but this did not occur with TIP49. The double hexameric complex of TIP48/TIP49 was also analysed and indicated a heterodisperse population of species, which provided clues about assembly pathways. TIP48 and TIP49 are structurally homologous to the bacterial branch migration motor RuvB. TIP48 and TIP49 do not incorporate the DNA binding domain present in RuvB, and instead have acquired a novel domain (Domain II) inserted between the Walker A and B ATPase motifs. Using mutants, we demonstrated that TIP48 Domain II is critical for regulating nucleotide dependent hexamerisation of TIP48. Furthermore, TIP48/TIP49 complexes lacking Domain II indicated that hexamer-hexamer interactions are not exclusively mediated by Domain II; this domain may regulate the assembly of different forms of the heteromeric complex. Interactions between TIP48 and TIP49 and several putative binding partners were tested in vitro. The ATPase activity and oligomerisation of TIP48 and TIP49 were assayed in the presence of Hint1, which was reported to disrupt the homotypic and heterotypic interactions within the TIP48/TIP49 complex. Finally, the sub-cellular localisation and post translational modification of TIP48 and TIP49 were analysed. Using immunofluorescence and GFP-tagged TIP49, we confirmed that TIP48 and TIP49 associate with the mitotic spindle; however, TIP48 accumulates at the midbody during cytokinesis, while TIP49 does not. Furthermore, we investigated potential modifications of TIP48 and TIP49 by 2-D polyacrylamide gel electrophoresis, which may account for the regulation of these distinct functions.

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