Molecular analysis of vesicle biogenesis during autophagy
Molecular analysis of vesicle biogenesis during autophagy
von Sebastian Bremer
Datum der mündl. Prüfung:2009-05-29
Erschienen:2009-05-14
Betreuer:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Gerhard Braus
Dateien
Name:bremer.pdf
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Description:Dissertation
Zusammenfassung
Englisch
Autophagy in Saccharomyces cerevisiae comprises diverse processes that transport cytoplasm and even organelles into the vacuolar lumen for degradation. During macroautophagy out of the pre- autophagosomal structure (PAS) autophagosomes are formed that fuse with the vacuole for degradation. During microautophagy cargo is directly engulfed by the vacuolar membrane. One aim of this study was to identify membrane sources needed for the formation of autophagosomes out of the PAS. Atg9 a transmembrane protein has been proposed to cycle between the PAS and a peripheral pool that may serve as a membrane source. In S. cerevisiae this pool has been described to colocalize in part with mitochondria. This was questioned in mammalian cells where this peripheral pool colocalizes with the trans Golgi network and endosomes. Another suitable candidate to follow the membrane flow is Atg8 that is covalently coupled to membranes via phosphatidylethanolamine (PE). This study describes for the first time a peripheral pool of Atg8 located at endosomes. Its formation is dependent on Atg1 and Atg27 as described for the Atg9 peripheral pool. Atg8 is conjugated to PE at the peripheral pool. Micronucleophagy (piecemeal microautophagy of the nucleus; PMN) occurs at nucleus-vacuole (NV) junctions and results in the pinching-off and release of nonessential portions of the nucleus into the vacuole. In contrast to previous published results Krick et al. showed in a recent publication that PMN requires the core macroautophagy genes. They analysed the degradation of the PMN marker protein GFP-Osh1 by quantifying the release of hydrolase resistant free GFP in the vacuole using immuno-western blot analysis. In this study a microscopic assay was established which supports the finding that PMN is efficiently inhibited in atg mutant cells. A nuclear resident fluorescent protein (NLS-mCherry) is used as marker protein. Part of this fluorescent protein pinch-off the nucleus dependent on the autophagic machinery and is degraded in the vacuole. The release of PMN vesicles at the NV-junctions requires at least three membrane fusion events. Krick et al. reported that the standard homotypic vacuolar fusion machinery is not required for the formation of PMN vesicles. This study indicates the requirement of Cdc48 and its major substrate-recruiting factor Shp1 for efficient PMN Summary 2 as well as macroautophagy. The human homologues of Cdc48 / Shp1 p97 / p47 are involved in human mitotic Golgi reassembly. This p97 / p47 complex is thought to extract a monoubiquitylated fusion inhibitor out of membranes and thereby mediate fusion. In this study the involvement of monoubiquitin in PMN as well as macroautophagy could be shown in S. cerevisiae. The ubiquitin deconjugation enzyme Ubp3 and its cofactor Bre5 are also required for PMN but not for the Cvt-pathway or autophagy. The vps class E genes vps27 and vps28 are also exclusively required for PMN. So far the fusion of the edges of double-membrane structures leading to autophagosomes was dubious, since neither NSF / Sec18 nor t-SNARES are required. Therefore a novel membrane fusion machinery requiring the Atg proteins has been proposed. In contrast this work supports the function of the previously excluded AAA+ ATPase Cdc48 in autophagic membrane fusion events.
Keywords: yeast S.cerevisiae starvation PAS autophagy; yeast S.cerevisiae starvation PAS autophagy