New Insights into Nucleophagy in S. cerevisiae
von Stefanie Karnebeck
Datum der mündl. Prüfung:2019-03-11
Betreuer:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Blanche Schwappach
Gutachter:Prof. Dr. Stefan Jakobs
EnglischNucleophagy is a specific type of autophagy, a process for the degradation of non-essential parts of the nucleus under starvation conditions. Nucleophagy can be subdivided into Atg39 and Atg40 dependent macronucleophagy (Mochida et al., 2015) and piecemeal microautophagy of the nucleus (PMN), also called micronucleophagy (Roberts et al., 2003). PMN takes place at the contact site between nucleus and vacuole, the nucleus-vacuole junction (NVJ) (Roberts et al., 2003). The backbone of the NVJs is formed by the interaction of Nvj1 and Vac8 (Pan et al., 2000a). Furthermore, Osh1 and Tsc13 are part of this cellular contact site (Kohlwein et al., 2001; Levine and Munro, 2001). Surprisingly, within this study it was shown, that degradation of GFP-Osh1, a marker protein for PMN, can be degraded independent of Nvj1. Recently, the structure of Osh1 was partially solved and it was shown, that a mutated version of Osh1, Osh1_A159V, is impeded in its interaction with Nvj1 (Jeong et al., 2017). Comparison of GFP-Osh1 and GFP-Osh1_A159V degradation showed equal results for both constructs in different deletion strains. This suggests, that impeded interaction of Osh1 and Nvj1 or the absence of NVJs results in an autophagic degradation of GFP-Osh1 different from PMN. Therefore, Nvj1-GFP is considered to be a better marker for PMN measurement. However, a genetic negative control for PMN isn’t available at the moment. Also, a truncated version of Nvj1, Nvj1∆318-321, that is known to bind to Vac8 and impedes Vac8 dimerization, but still forms proper NVJs (Jeong et al., 2017) can not serve as negative control for PMN measurement. This construct was shown to localize to the perinuclear ER and was degraded to a greater extent as Nvj1-GFP via Atg39 dependent macronucleophagy. The N-terminus of Nvj1 is known to be essential for strict localization to the perinuclear ER (Millen et al., 2008). In this study it was shown, that the hydrophobic core of the N-terminus is crucial for the degradation of Nvj1. Therewith, this marker protein can be used as negative control for PMN measurement. This suggests a role of the hydrophobic core of Nvj1 in the bulging of the NVJ into the vacuole and therewith PMN/ NVJ-phagy. Degradation of Nvj1-GFP was completely blocked in atg39∆cells. Therewith, this study showed for the first time, that Atg39 is not only essential for macronucleophagy (Mochida et al., 2015), but also plays an important role in PMN. Due to the lack of negative control for PMN measurement, several genes were tested in regard to their involvement in PMN. All tested genes, including NVJ2, SCS2, GLC8 and YMR310C were shown to be dispensable for PMN. However, in scs2∆ cells the NVJs appeared to be affected. Degradation of the nucleolar marker Nop1-GFP was shown to be independent of Nvj1 and therewith PMN. This means, that Nop1-GFP is mainly degraded via Atg39 dependent macronucleophagy (this study) (Mochida et al., 2015). Based on these results is seemed to be reasonable that the main goal of PMN is not the degradation of nuclear material, but the degradation of the NVJ together with the associated protein machinery. Therefore, PMN should be renamed into NVJ-phagy. Finally, the two marker proteins Nab-NLS-mCherry and Pho8-GFP were tested in regard to their ability for measuring of PMN/ NVJ-phagy or the differentiation between micro- and macroautophagy, respectively. Unfortunately, both marker proteins appeared not to be suitable for the intended purpose.
Keywords: Autophagy; S. cerevisiae; NVJ-phagy; Nucleophagy