| dc.description.abstracteng | Macroautophagy is a degradative membrane trafficking pathway that removes and recycles cellular components to maintain cellular homeostasis. Targets for degradation are a variety of cytosolic components including protein aggregates, damaged or superfluous organelles or invading pathogens. The transport process can either be unselective or selective but always involves the sequestration of a cargo by a double-membraned vesicle, the autophagosome. Upon starvation the autophagosome biogenesis is initiated at the pre autophagosomal structure (PAS) where a cup-shaped membrane structure is assembled de novo (phagophore) to engulf cytosolic material. After closure, the autophagosome fuses with the vacuole to release its cargo to the vacuolar lumen for degradation and reuse.
A crucial factor for autophagosome biogenesis is the generation of phosphatidylinositol 3-phosphate (PI3P) at the PAS. The presence of PI3P on autophagic membranes is deciphered by β-propellers that bind polyphosphoinositides (PROPPIN). PROPPINs are a highly conserved family of WD40-repeat proteins which fold as seven-bladed β-propellers. PI3P binding is mediated by a conserved FRRG-motif that participates in the formation of two lipid-binding sites at the circumference of the propeller.
Another important step required for the elongation of the phagophore, is the conjugation of ubiquitin-like Atg8 to the lipid phosphatidylethanolamine (PE) which is mediated by two ubiquitin-like conjugation systems. The first system results in the coupling of ubiquitin-like Atg12 to Atg5. In complex with Atg16, the Atg12-Atg5 conjugate acts as an E3-like enzyme to facilitate the transfer of Atg8 from its E2-like enzyme Atg3 to PE.
This study focused on the investigation of the molecular functions of the S. cerevisiae PROPPIN Atg21. So far, the only known function of Atg21 is to organize the lipidation of Atg8 at the PI3P-positive autophagic membrane. It recruits the Atg8-Atg3 conjugate and the Atg12-Atg5/Atg16 complex to the PAS by a direct interaction with Atg8 and Atg16. Unlike its homolog Atg18, Atg21 is not essential for bulk autophagy but is indispensable for some selective types of autophagy including the Cytosol-to-vacuole targeting (Cvt) pathway. The Cvt-pathway selectively transports proaminopeptidase 1 (pApe1) and other hydrolases to the vacuole under nutrient rich conditions.
In parallel to this study the cooperation partners Dr. Karin Kühnel and Dr. Janina Metje were able to solve the crystal structure of Kluyveromyces lactis Atg21 in complex with the coiled-coil domain of Ashbya gossypii Atg16 up to a resolution of 4 Å. This is the first crystal structure of a PROPPIN-family member in complex with its interactor. On the basis of the crystal structure, a detailed analysis of the interacting domains of Atg21 and Atg16 was performed in this study. A variety of in vivo and in vitro experiments revealed that Atg21 and Atg16 interact through the formation of a single salt bridge between Atg21 R151 and Atg16 D101. This salt bridge is essential for the proper targeting of the Atg12-Atg5/Atg16 complex to the PAS as well as for an efficient progression of the Cvt pathway. Surrounding amino acids seem to stabilize the salt bridge by contributing to the net charge on the surface of both proteins but are dispensable for their interaction and function. This interaction mechanism of Atg21 and Atg16 is conserved from yeast to mammals.
Fluorescence cross-correlation spectroscopy (FCCS) in combination with other approaches indicated that the Atg21-Atg16 complex might be preferentially assembled at the autophagic membrane and not in the cytosol.
Overexpression of APE1, the cargo of the Cvt-pathway, results into the formation of an artificially enlarged cargo complex which enables the monitoring of phagophore assembly and elongation by fluorescence microscopy (APE1-overexpression assay). In this study, using the APE1-overexpression assay, a yet unknown contact site between the vacuole and the phagophore was identified. The contact site is established by the vacuolar protein Vac8 and is important for the elongation of the phagophore. Although Vac8 seems to be not essential for the assembly of the PAS, it defines the normal site of its formation. The vacuole-phagophore contact site coincides with the formation of a specialized membrane domain at the vacuolar membrane. This membrane domain is characterized by the enrichment of Vac8 and the exclusion of the membrane integral protein Vph1. This suggests the formation of a lipid raft at the vacuolar membrane although the contribution of lipids to the membrane domain requires further investigation.
Under starvation conditions the localization of Atg21 at the phagophore is restricted to the contact site between vacuole and phagophore. Thereby, Atg21 targets the Atg8 lipidation to the highly curved rim of the growing phagophore. Atg21 is not essential for the formation of the vacuole-phagophore contact site. Nevertheless, it might contribute to it as a potential interactor of Vac8.
Similar to Atg21, its homolog Atg18 also resides on the rim of the phagophore although PI3P is dispersed over the whole phagophore. Therefore, there must be additional mechanisms that regulate the localization of PROPPINs at the phagophore. This could include the sensing of membrane curvature or even the induction of membrane bending.
PROPPINs localize to various organelles in the cell (endosomes, vacuole and autophagic membrane) suggesting that they exhibit a variety of functions. Nevertheless, very little is known about their other functions. In this study, the proximity dependent biotin identification (BioID) assay in combination with a stable isotope labeling by amino acids (SILAC) based approach was used to quantitatively identify potential interactors of Atg21. In an initial experiment 17 potential interaction partners of Atg21 were identified. The majority of the identified proteins can be characterized as membrane associated proteins which reside on vacuolar and endosomal membranes or on cytosolic vesicles. Functionally most of them participate in transport processes, mainly vesicle-mediated transport processes. The localization and function of the potential interactors of Atg21 highly correlates with the localization of Atg21. Therefore, the 17 potential interaction partners of Atg21 provide a promising basis for the elucidation of the role of Atg21 in the cell. | de |