| dc.description.abstracteng | Autophagy summarizes a family of intracellular lysosomal (vacuolar) degradative transport process. It is used for the removal and recycling of cytoplasm, protein aggregates and organelles to maintain cellular homeostasis. During macroautophagy in yeast, the double membrane layered transport vesicles (autophagosomes) are formed at the pre-autophagosomal structure (PAS). They finally fuse with the vacuole for degradation and recycling of their contents. Beside this unselective degradation of bulk cytoplasm including organelles, there are several selective types of autophagy. In yeast, the Cytoplasm-to-vacuole (Cvt) pathway selectively transports the hydrolases α-mannosidase, proaminopeptidase I and aspartyl aminopeptidase to the vacuole under nutrient rich conditions.
A hallmark of autophagy is the use of two ubiquitin-like conjugation systems. One covalently couples the ubiquitin-like Atg12 to Atg5. The resulting Atg12-Atg5 conjugate forms a complex with Atg16 that acts as an E3-like enzyme in the second ubiquitin-like conjugation reaction, which couples Atg8 to the head group of the membrane lipid phosphatidylethanolamine (PE). Atg16 specifies the lipidation site, but how it selectively recognizes and binds to the PI3P-positive PAS remained unclear.
In this study, the localization and function of Atg21 was investigated using the model organism Saccharomyces cerevisiae. Atg21 forms a WD40 propeller that is peripherally membrane associated by binding to PI3P and PI(3,5)P2. So far, Atg21 has only been detected at endosomal compartments and at vertices of vacuolar junctions. Atg21 is required for selective types of autophagy as the Cvt pathway and efficient bulk macroautophagy. In the absence of Atg21 no Cvt vesicles are formed, suggesting a fundamental role for Atg21 in the PAS assembly especially during selective types of autophagy. Indeed, growing atg21∆ cells fail to efficiently lipidate Atg8 and to recruit Atg8 and Atg5 to the PAS. Since Atg21 has not been detected at the PAS so far, its precise molecular function during autophagy remained elusive.
Using direct fluorescence microscopy, this study revealed, for the first time, that part of Atg21 is also present at the PAS. The presence of Atg21 at the PAS is dependent on Atg14 and therefore on the PAS-specific PI3P pool. Moreover, it could be shown in this study, that Atg21 recruits the Atg12-Atg5/Atg16 complex to the PAS via direct interaction with Atg16. Two conserved surface residues within the coiled-coil domain of Atg16 (D101 and E102) were identified to specifically bind Atg21. Thereby the underlying mechanism of Atg16-mediated recruitment of the Atg12-Atg5/Atg16 complex to autophagosomal membranes has been elucidated in this study. More importantly, it was shown, that the interaction of Atg21 and Atg16 was not only a prerequisite for the proper PAS localization of the Atg12-Atg5/Atg16 complex but also for its autophagic function. Consequently, interaction of Atg16 and Atg21 is needed for efficient Atg8 lipidation.
Importantly, this study also revealed that Atg21 and Atg8 probably interact with each other. This interaction occurred with unlipidated Atg8, suggesting, that Atg21 not only mediates the membrane association of the E3 enzyme Atg12-Atg5/Atg16, but also acts as a scaffold for Atg8 lipidation reaction. Therefore, Atg21 represents the missing link that mediates PI3P-dependent PAS association of the lipidation machinery by recruiting the activated substrate Atg8 and the E3 enzyme required for the transfer of Atg8 to PE. Atg21 thus represents a key factor to specify the lipidation site. This model provides a reasonable explanation for the elementary function of Atg21 in Cvt vesicle formation.
By using gelfiltration chromatography and sorbitol gradient density centrifugation Atg21 was identified as part of a high molecular weight complex. This Atg21 complex is very sensitive to the cell lysis procedure and buffer conditions. Therefore, a mild cell lysis procedure was established to keep the sensible complex intact as a prerequisite not only for further analysis of the complex but also for the identification and verification of additional potential Atg21 interacting proteins.
Pull down experiments and following mass spectrometry analysis identified the target of rapamycin complex I (TORC1) as potential interaction partner of Atg21. It is already known, that the kinase activity of TORC1 negatively regulates autophagy.
A specific interaction of Atg21 with the type I membrane protein Atg27 but with no other member of the trimeric Atg27-Atg9-Atg23 complex was detected using the split-ubiquitin system. Moreover, it could be suggested that Atg21 interacted with the cytosol-facing C-terminal part of Atg27. In addition, using direct fluorescent microscopy it could be shown here that a considerable part of Atg21 colocalized with Atg27, supporting a potential interaction. The relevance of this putative interaction will be topic of further studies. | de |