Dissecting molecular elements of mitophagy and the lysis of intravacuolar vesicles

Autophagy is an evolutionary conserved catabolic process essential for the recycling and removal of cytosolic components via the lysosome (vacuole). Macroautophagy is characterized by the formation of a double membrane-layered autophagosome. Autophagosome biogenesis starts at the preautophagosomal structure (PAS). Here, the phagophore, a cup-shaped double membrane-layered structure is generated, which expands around its cytosolic cargo and finally fuses at its edges to form an autophagosome. Subsequently, the outer autophagosomal membrane fuses with the vacuole to release the inner monolayered autophagic body into the vacuolar lumen for its degradation. Mitophagy as a selective variant of autophagy is crucial for removal of damaged or superfluous mitochondria. This quality control mechanism is necessary because mitochondria produce ROS as a by-product while generating ATP. Atg32 is the receptor for mitophagy and was analyzed in this thesis. The carboxyterminus of Atg32 is exposed to the mitochondrial intermembrane space. The lack of this region impaired mitophagy drastically, which could be based on its importance for proper mitochondrial localization. Furthermore, under strong mitophagy inducing conditions, overexpression of Atg32 led to a disturbed mitophagy rate, which was again dependent of the IMS domain. Deletion of the whole Atg32 gene revealed a potential second function in cell homeostasis during ROS stress. atg32∆ cells showed a strongly decreased growth rate on H2O2 containing plates and increased respiration. Unfortunately, the induction of mitophagy via ROS stressors such as H2O2 or paraquat led only to a very faint mitophagy induction. The established method for monitoring mitophagy required the cultivation in non-fermentable lactate medium. This method was modified to perform a large-scale screen for mitophagy defects in a library of deletion strains unable to grow on lactate. Large-scale analysis led to no clear results and therefore, several specific deletion strains were analyzed. Because of contradictory published data, the importance of Uth1 for mitophagy was reanalyzed and demonstrated its dispensability for mitophagy. Furthermore, the increased mitophagy-dependent degradation of damaged mitochondria was verified in YME1 deleted cells. The ESCRT complexes II and III showed a severe reduction of mitophagy, whereas deletion strains of ERMES and the RTG pathway were unaffected. After import of autophagic bodies or MVB vesicles to the vacuolar lumen, these vesicles have to be lysed before the cargo can be degraded. Atg15 contains a lipase-active motif and is essential for these lysis events in the vacuole. In the second part of this thesis, the localization of endogenously expressed Atg15 was analyzed. For the first time, Atg15 was visualized at autophagic bodies and at the PAS. This is remarkable because Atg15 is the first identified integral membrane protein of mature autophagosomes. Atg15 was further located at the ER and ER exit sites. ER exit sites are linked to phagophore elongation and could therefore represent an entry point of Atg15 into the autophagosomal pathway. An almost complete colocalization of Atg15 and prApe1 in several autophagy-deficient deletion strains further prompted the hypothesis that Atg15 and prApe1 get in contact before PAS assembly at the vacuolar membrane. In contrast to previous assumptions, determination of Atg15 topology showed that Atg15 only contains one N-terminal TMD with its N-terminus in the cytosol and the C-terminus in the ER lumen. Deletion of the TMD reduced its biological activity and ability to enter the ER lumen. This demonstrates the function of the TM region especially for ER import and subsequent transport to the vacuole. Atg15 truncated for its TMD showed characteristics of a peripheral membrane associated protein. The lipase activity of Atg15 in the vacuolar lumen was essential for degradation of MVB vesicles, ABs and Atg15 itself. Therefore, this study suggests that Atg15, which is localized in the membrane of these vesicles, degrades its embedding membrane and allows subsequent degradation of the cargo. Here, the peripheral membrane association brings the lipase active site in close proximity to the membrane destined for degradation. How Atg15 is activated exclusively in the vacuolar lumen is still elusive. The overexpression of active, membrane-bound Atg15 variants reduced cell growth in different deletion strains. This was still the case in some mutants of the v-ATPase or different transport pathway. It is thus unlikely that the vacuolar pH or a high substrate specificity of Atg15 is responsible for its activation. Based on sequence homology, Atg15 was identified as a α/ß-hydrolase with a potential catalytic lid, which could be controlled by a yet unknown additional component such as a colipase.