Dissecting the Atg21 organization at the growing phagophore and the evolutionary conservation of Nvj1 among yeasts
by Florian Kramer
Date of Examination:2024-11-14
Date of issue:2024-12-09
Advisor:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Silvio Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-10932
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Abstract
English
The first project of this thesis deals with the spatial and temporal regulation of Atg21 at growing phagophores. Macroautophagy is a highly conserved process involved in the degradation and recycling of cellular material reaching from cytoplasmic material to entire defective or superfluous organelles. The main proteins facilitating this process are 42 Atg proteins, that are recruited to the PAS in a highly regulated, hierarchical order. After the initial induction of macroautophagy by the Atg1 kinase complex and the subsequent formation of an isolation membrane, generated from Atg9-rich vesicles, the PI3-kinase complex I is recruited to this membrane. Contained in the PI3-kinase complex is Atg14, which is responsible for the recruitment of the complex to the PAS and also upholds an interaction with the vacuolar protein Vac8, which confines the complex to the vacuolar tethered edge of the isolation membrane. Here the complex is involved in the phosphorylation of phosphatidylinositol (PI) to phosphatidylinositol 3-phosphate (PI3P). The Atg18-Atg2-Atg9 complex further tethers the other side of the isolation membrane to the ER. For the following elongation of this isolation membrane, it is necessary to conjugate the protein Atg8 with phosphatidylethanolamine in the isolation membrane. A major contributor to this process is the β-propeller protein Atg21, which acts as a protein hub and recruits the complexes involved in Atg8 lipidation. As part of the PROPPIN protein family, Atg21 can bind the newly produced PI3P in the isolation membrane. This lipid binding is necessary for its localization, but not sufficient to explain its narrow recruitment to only the vacuolar edge of the isolation membrane. This thesis aimed to examine possible candidates that could provide a concentrated localization of Atg21 to this region. Atg14 was quickly identified as a prime contender since it localizes the PI3-kinase complex I to the vacuolar edge and like Atg38, is only found in the autophagic PI3-kinase, while the other proteins of this complex also localize to endosomes. After initial positive results that demonstrated co-isolation of Atg14 and Atg21 in Co-IPs, it was later shown, that this interaction is at least partially dependent on Vps15. More sensitive methods like the recombinant expression and co-isolation of these proteins as well as a split ubiquitin approach, were not successful in showing a direct interaction between Atg14 and Atg21. Furthermore, efforts were made to narrow down possible binding regions in Atg14, which require further investigation. The second project included in this thesis deals with the nucleus vacuole junction (NVJ), which is a membrane contact site (MCS) established by the binding of Nvj1 in the outer nuclear membrane with Vac8 in the vacuolar membrane. In its function as an MCS, it promotes lipid biosynthesis and the non-vesicular lipid transport between the membranes, by recruiting multiple proteins involved in these processes. Thereby it contributes to the overall lipid homeostasis in the cell and the formation of membrane subdomains, important for a multitude of processes. The other function of the NVJ lies in the autophagic process of piecemeal microautophagy of the nucleus (PMN), which is induced by nitrogen starvation and leads to the teardrop-shaped invagination of the NVJ into the vacuole, containing nuclear material and the junction itself, together with all previously localized proteins. This structure finally buds off and is degraded. However, the PMN process failed to show any significant effect on cell survivability under standard starvation conditions, which stands in stark contrast to the macroautophagic mechanism. Additionally, the Nvj1 protein consists of a small number of binding domains, connected by a mostly disordered linker region, which is very permissive for mutations. Both the low impact on survivability and the disordered structure could have allowed for an extremely divergent evolution of this protein and led to the hypothesis that Nvj1 is a lineage-specific gene for a small group of close relatives to the Saccharomyces genus. In this thesis, micro-synteny analysis was used to expand on identified Nvj1 orthologs and to examine the possible functional conservation of its roles in NVJ establishment. Multiple positional orthologs, that diverged from S. cerevisiae over 200 million years ago and retain no detectable sequence similarity, were found to engage in the formation of the NVJ, when endogenously expressed or even in S. cerevisiae. Furthermore, it was demonstrated that Nvj1 proteins from distant relatives were still able to facilitate PMN and also partially recruit proteins of the lipid biosynthesis machinery, thereby hinting strongly at a possible functional conservation of these processes in these divergent species. The access to far more, newly identified sequences for Nvj1 homologs, also gave the opportunity to identify a previously elusive, well-conserved motif in the Nvj1 sequence, that evolutionary was bound to the Osh1 binding site of Nvj1. The now-termed Osh1-associated motif, was subsequently mutated and efforts were made to describe the mutant phenotype and identify an interaction partner of this motif.
Keywords: Autophagy; Atg21; Nvj1; PMN; Vac8; NVJ; Piecemeal Microautophagy of the Nucleus; Nucleus-Vacuole Junction
