| dc.description.abstracteng | Autophagy is a highly regulated cellular degradation and recycling process conserved from yeast to higher eukaryotes. This process is constitutively active at a low level, and non-selectively degrades portions of the cytosol in response to internal or external stimuli. This process begins with the de novo formation of a cup-shaped membrane structure at the pre-autophagosomal structure (PAS) termed the isolation membrane or phagophore. The phagophore expands to fully engulf the cargo before closing to form the double-membrane structure known as the autophagosome which then fuses with the vacuole to release its cargo into the vacuolar lumen for degradation and recycling.
The biogenesis of the autophagosomes requires the generation of phosphatidylinositol 3-phosphate (PI3P) at the PAS. The presence of PI3P at the PAS allows for the binding of -propellers that bind polyphosphoinositides (PROPPIN). The PROPPINs are a highly conserved family of WD40-repeat proteins and structurally fold as seven-bladed -propellers. WD40 domains are key components of proteins that mediate protein-protein interactions, including scaffolding, cooperative assembly, and regulation of dynamic multi-subunit complexes. PROPPINs also have a conserved FRRG-motif at the circumference of the propeller that allows for binding phosphoinositides.
In Saccharomyces cerevisiae, there are three PROPPINs: Atg18, Atg21 and Hsv2. They are highly homologous but have different autophagic subtypes specificities. Atg18 is a core autophagy protein required for all autophagy in a PI3P-dependent manner. Atg18 has a secondary role at the vacuole binding to PI(3,5)P2, where it carries out nonautophagic functions in regulating vacuolar morphology. Atg21 is not essential for unselective autophagy but is crucial for selective types of autophagy, including the Cvt pathway, which targets vacuolar hydrolases like prApe1 to the vacuole. Hsv2 is the least functionally characterised PROPPIN which is required for efficient piecemeal microautophagy of the nucleus. So far, this is the only observation that has been seen to affect autophagic activity.
This study focused on investigating the molecular function of the S. cerevisiae Hsv2. As very little is known about the role of Hsv2, a proximity-dependent biotin identification assay in combination with a stable isotope labelling by amino acids based approach was used to identify potential interactors of Hsv2. In this approach, 50 proteins were identified as potential interactors of Hsv2. To confirm these interactions, Hsv2-GFP and identified proteins tagged with HA were tested for coimmunoprecipitation using a GFP-Trap. Yck3, Vtc3, Vps35 and Vac14 are all confirmed interaction partners of Hsv2 using these methods. Many of these proteins localise to the vacuolar membrane which correlates with the localisation of Hsv2. In parallel, a split ubiquitin assay was also used to further the understanding of the Hsv2 interactome. Using this method we saw an interaction between Hsv2 and Vps21, Sso1, Pep12, Tlg1, and Snc1. These potential interacting partners of Hsv2 provide a promising basis for elucidating the role of Hsv2 within the cell.
In addition, we determined the potential interaction site between Atg21 and Atg8. By purifying Saccharomyces cerevisiae Atg8 and with the subsequent binding assays with a synthetic peptide of Atg21, we found the binding affinity to be 42.5 nM. Once the binding affinity had been determined, crystallisation trials were initiated to deduce the crystal structure of Kluyveromyces lactis Atg21 in a complex with ScAtg8. Crystals that formed were tested using X-ray crystallography, these were found to be formed of salt and not protein. Computational modelling of the known crystal structures of KlAtg21 and ScAtg8 confirmed that residue D146 of Atg21 could be essential for binding to Atg8.
Finally, part of this study was to dissect the functional role of Atg18 with the retromer complex in regulating vacuolar morphology. Using hyperosmotic stress to induce vacuolar fragmentation, we found that Vps35, Vps29 and Vps26 are required for efficient Atg18 fission activity at the vacuolar membrane. Additionally, in the absence of Vps5 and Vps17, there was unregulated fission activity at the vacuolar membrane that was not dependent on hyperosmotic conditions. These results suggest the importance of Vps35, Vps29 and Vps26 in the efficient fission activity of Atg18 as well as the value of Vps5 and Vps17 in impeding the fission activity of Atg18. | de |