Analyse der molekularen Funktionen des Ubiquitin-artigen Atg8 bei der Autophagosomen-Biogenese in S. cerevisiae
Zusammenfassung der Projektergebnisse
Autophagy is a highly conserved intracellular vesicle transport pathway that avoids the accumulation of harmful material within cells. The formation of the unique double membrane layered transport vesicles, the autophagsosomes, is initiated at the socalled phagophore assembly site (PAS). Here, the autophagic proteins localize at least transiently and their dynamic assembly and disassembly of the different protein complexes is strictly regulated. In general, Rab GTPases are major regulators of cellular vesicle trafficking. They act as molecular switches in membrane trafficking. Rab GTPases are activated by guanine-nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). We identified a GTPase-activating protein (GAP) as novel interaction partner of the ubiquitin-like key component of autophagy Atg8. Interestingly, the identified GAP stimulates the hydrolysis of GTP on the Rab GTPase Ypt1, that has been already associated with autophagy, just like its GEF, TRAPPIII. Detailed analysis revealed that the GAP localizes to the PAS under nutrient rich conditions and its GAP activity is required for selective autophagic variants, such as the Cvt pathway, that acts under nutrient rich conditions, or the selective autophagic degradation of mitochondria (mitophagy). Using fluorescence microscopy and co-immunoprecipitation assays, we could identify the step during PAS assembly that requires this GAP activity. It has been already shown that efficient interaction of Ypt1 with its effector Atg1 requires active GTP-bound Ypt1. We could now show that the dynamic disassembly of the conserved Ypt1-Atg1 complex is regulated by the GAP activity under nutrient rich conditions. The efficient disassembly of the Ypt1-Atg1 complex is a prerequisite for the recruitment of Atg14 to the PAS, leading to production of phosphatidylinositol 3-phosphate (PtdIns3P). Under nutrient rich conditions PtdIns3P is decoded by the PROPPIN Atg21 that organizes the conjugation of Atg8 to the membrane lipid phosphatidylethanolamin (PE). In its membrane-associated form Atg8, among other functions, tethers the receptor-bound cargo complexes to the inside of the phagophore. Interestingly, efficient formation of the cargo receptor-Atg8 complexes requires the interaction of the GAP with Atg8. Taken together, the identified GAP is required for 2 separate steps of PAS assembly during selective autophagy. It acts as a GAP of Ypt1 to mediate efficient disassembly of the Ypt1-Atg1 complex and later as an Atg8 interaction partner. These findings clarify one molecular mechanism of complex disassembly during phagophore formation and suggest that GAPs might have dual functions in cellular vesicle trafficking.
Projektbezogene Publikationen (Auswahl)
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(2019) Gyp1 has a dual function as Ypt1 GAP and interaction partner of Atg8 in selective autophagy. Autophagy 15 (6) 1031–1050
Mitter, Anne Lisa; Schlotterhose, Petra; Krick, Roswitha
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“Dissecting the molecular functions of the ubiquitin-like Atg8 during autophagosome biogenesis in S. cerevisiae.” December 2017, Goettingen
Anne Lisa Mitter
