Genetics - Theses
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ItemThe mammalian stonin proteins : from translation to degradation(University of Melbourne, 2009)Recent work has centred around the role of stonin 2 in clathrin-mediated endocytosis (CME) of the integral synaptic vesicle protein synaptotagmin I. Without a mode of internalisation of this protein, synaptic vesicles that are formed from the plasma membrane are fusion-incompetent and therefore no neurotransmitter can be released (Mohrmann, 2008). Stonin 2 was first identified on the basis of homology with the Stoned B gene of Drosophila melanogaster. The stoned B protein is encoded in the second open reading frame (ORF) of a dicistronic transcript that encodes another protein involved at the synapse, named stoned A. It was in 1973 that the locus was first identified in a screen for temperature sensitive paralytic mutants. These mutants were shown to have defective nervous systems and had aberrant synaptic vesicle retrieval at the plasma membrane of the neuromuscular junction (NMJ). Like stonin 2, stoned B interacts with synaptotagmin I through its C-terminal domain that shows significant homology to the ?2 subunit of the clathrin adaptor protein complex AP-2. Stonin 2 is not the only stoned B homologue in mammalian genomes, with another protein named stonin 1 showing equal protein similarity. In this report, the stonin 1 antibodies that had previously been utilised (Arnott 2004) were re-characterised and it was concluded that they were only useful to detect expressed recombinant protein. Affinity purified stonin 2 antibodies, created in this project, were successfully utilised to detect both endogenous stonin 2 and recombinant expressed-tagged stonin 2 for both Western blot and immunocytochemistry, which required a novel antibody purification method to be created to achieve a greater resolution. After analysis of expressed tagged stonin proteins in the neuroendocrine PC12 cell line, it was suspected that both proteins were being rapidly degraded. This led to the discovery that both stonin 1 and 2 were being targeted for degradation by the ubiquitin proteasome system that appears to be neuroendocrine specific. Stonin 2 is additionally cleaved by an unknown protease to produce C-terminal fragments that are the predominant protein species in PC12 cells. The analysis of the stonin 2 protein fragmentation pattern compared to the endogenous banding pattern led to the discovery that protein variation is also achieved through initiation of translation at internal start sites. Although the specific methionine residues were not characterised, at least two protein species were identified that were the result of internal initiation. These protein species may be regulated through the use of a short upstream open reading frame that overlaps the predicted stonin 2 ORF start codon. The internal start sites for translation initiation would give rise to N-terminally truncated protein species that would lack one or two critical AP-2 binding motifs that may modulate the function of stonin 2. The final series of experiments presented in this thesis analyse the localisation and protein-protein interactions of the stonin proteins through immunocytochemical analysis of exogenously expressed-tagged stonin proteins during inhibition of the ubiquitin proteasome system. This lead to the finding that when stonin 1 is bound to internal membranes it is predominantly bound to internal structures that can be motile. Under these conditions, it was also shown that stonin 1 could bind synaptotagmin I, although it is unlikely to occur at the plasma membrane. Membrane-bound Stonin 2 is found predominantly sub-cortically or at the plasma membrane. The sub-cortical localisation may be at sites that vesicles are, or will be, attached to the plasma membrane. Stonin 2 can be part of a protein complex that involves AP-2, synaptotagmin I and the fission molecule dynamin I. These results provide new insight into the role that both stonin 1 and 2 play in the cellular trafficking network. The protein variation identified in Stonin 2 adds further complexity to the myriad of regulatory events that can occur to modulate synaptic plasticity.