Identification of novel components involved in selective and unselective autophagic pathways
Identifizierung neuartiger an selektiver und unselektiver Autophagy beteiligter Komponenten
von Evelyn Welter
Datum der mündl. Prüfung:2011-05-16
Erschienen:2012-04-11
Betreuer:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Peter Rehling
Gutachter:Prof. Dr. Gerhard Braus
Gutachter:Prof. Dr. Ralf Ficner
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-3159
Dateien
Name:welter.pdf
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Zusammenfassung
Englisch
Autophagy is a ubiquitous protein degradation pathway in eukaryotic cells. As response upon nutrient starvation, cytosolic material and also whole organelles are engulfed by a double membrane-layered vesicle, the autophagosome. The formation of the autophagosome starts at the pre-autophagosomal structure (PAS). The completed autophagosome is then targeted to the vacuole. After fusion with the vacuole, a single membrane-layered vesicle, the autophagic body, is released into the vacuole. After lysis of the membrane, the contents of the autophagic body are degraded and the contained macromolecules are recycled. The aim of this study was the identification of cooperating or redundant genes to different autophagic genes, to uncover molecular details of autophagosome formation and to allow mechanistic insights into different selective variants of autophagy. For the identification of autophagy related cooperating or redundant genes, the Synthetic Genetic Array analysis (SGA) was used. This is a powerful high-throughput technique to identify synthetic lethal double deletions, probably reflecting the same essential function of the respective gene products. For this purpose, the query strains atg8∆, atg15∆, atg18∆ and atg21∆ were generated in this study and crossed to the entire deletion library available for S. cerevisiae. Unfortunately, the procedure resulted in only false positive matches. In addition to the common marker protein GFP-Atg8, this study introduces the marker protein Pgk1-GFP to detect unselective autophagy by western blot and microscopy. Compared to the autophagic substrate GFP-Atg8, the use of this cytosolic protein allows the unbiased detection of unselective bulk autophagy. The molecular details of the biogenesis of autophagosomes are poorly understood. Our group identified the AAA+ ATPase Cdc48 and its cofactor Ubx1 (Shp1) as novel components needed for autophagosome biogenesis. In accordance with former experiments in the laboratory, this study presents Shp1 as the exclusive cofactor of Cdc48 needed for autophagy. SHP1 deleted cells show no accumulation of autophagosomes in the vacuole, while the formation of the PAS or the lipidation of Atg8, which both are important steps during autophagosome formation, are not impaired in shp1∆ cells. Furthermore, a suggested interaction of Shp1 and Atg8 was proven by recombinant pull down experiments using GST-Shp1 and some truncated versions. In mammals, the homologues of Cdc48 and Shp1, p97 and p47, are involved in the ubiquitin-dependent mitotic Golgi reassembly. In contrast, the complex of Cdc48 and Shp1 in yeast, requires the ubiquitin-like Atg8 for its function in autophagy. This gives for the first time a rationale for the use of Atg8 during unselective autophagy and explains why the delipidation of Atg8-PE by Atg4 is necessary for efficient autophagy. Mitochondria are the sites of ATP production in aerobic cells. As a consequence, they are at the same time a major source for reactive oxygen species (ROS), which damage mitochondria and other organelles. Thus, a quality control for mitochondria is essential for cellular survival. Mitophagy, the selective autophagic degradation of mitochondria, might fulfil such a role, but is still poorly understood in yeast. In this study, the construction of a marker protein and its use in degradation assay experiments by western blot allow the quantitative measurement of mitophagy. In contrast to a commonly used chromosomal Om45-GFP fusion, the novel marker protein can be expressed vom a plasmid, avoiding time consuming chromosomal integration. Different conditions to induce mitophagy were established. These different conditions allow the differentiation between mitophagy of superfluous or damaged mitochondria. Hereby, proteins of the ESCRT machinery, not required for unselective bulk autophagy, were found to be needed for mitophagy. In humans, Parkin ubiquitinates mitochondrial substrates to induce mitophagy. In this study, the ubiquitin isopeptidase Doa4 is shown to be required for mitophagy, indicating for the first time a mitophagy-related role of ubiquitin in yeast. Another selective variant of autophagy is PMN, the piecemeal microautophagy of the nucleus. A specific cargo-recognition in PMN is indispensable to preserve the integrity of the nucleus. Therefore, the still unknown nuclear cargo was a focus of interest. The results of this study point to a specific cargo-recognition of nucleolar proteins in PMN. Strains expressing different nucleolar GFP fusion proteins were investigated, some showed PMN-like structures in microscopy. Furthermore, nucleus-vacuole junctions, the contact sites between the nucleus and the vacuolar membrane, are shown to localise near the nucleolus.
Keywords: macroautophagy; PMN; mitophagy; Atg8; Pgk1-GFP
Schlagwörter: macroautophagy; PMN; mitophagy; Atg8; Pgk1-GFP
