Investigation of the ubiquitin proteasome system in Schizosaccharomyces pombe
Glover, James S. A.
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Ubiquitin is an essential 76 amino acid protein which can be conjugated to lysine residues on a variety of substrates via its C-terminal diglycine motif. This conjugation allows the protein to act as a molecular tag in a range of processes, including regulation of chromatin compaction, signalling cascades and DNA repair. In addition, ubiquitin moieties are capable of forming chains through the successive conjugation to lysine residues within ubiquitin itself. One of the most well characterized functions of ubiquitin is its role in protein quality control and degradation. Tetra-ubiquitin chains, most commonly through a lysine-48 linkage, are responsible for directing proteins to the 26S proteasome for degradation. This process is of importance both in the removal of miss-folded proteins, and in the regulated destruction of specific targets, such as the cyclins. The 90kDa AAA-ATPase Cdc48/p97/VCP is an essential protein that forms a hexameric complex, which interacts with a wide variety of ubiquitinated substrates. The specificity of Cdc48 is modulated by a series of different cofactors, which together allow Cdc48 to operate in several different contexts, from removal of misfolded proteins from the ER, to regulating securin stability. The role of two Cdc48 cofactors, Ubx4 and Ubx5, was studied in an attempt to dissect their function and to determine how they may modulate the function of Cdc48. Neither protein was found to be essential, as knockouts of either were found to be viable with no major defect in growth rate. The work also describes the findings of a yeast two-hybrid screen to identify potential substrates for both cofactors. Delivery of ubiquitinated proteins to the proteasome is mediated by shuttling factors, which are able to bind to both ubiquitin and the proteasome, and hence mediate the interaction between both. The shuttling factor Dph1 binds ubiquitin via a C-terminal UBA domain, while its N-terminal UBL domain mediates its interaction with the proteasome. This work identified a novel interaction between the Sti1 domains of Dph1 and the N-terminal region of a mitochondrial localized AAA-ATPase, homologous to the Saccaromyces cerevisiae protein Msp1. In addition, cell fractionation experiments revealed the presence of Dph1 at the mitochondria. This interaction provides hints that Mlp1 may be involved in the removal of ubiquitinated proteins from the mitochondria, and their delivery to the proteasome. The thesis begins to try and attempt to identify possible substrates of this proposed mitochondria associated degradation pathway, and looks for ways in which the hypothesis may be tested.