Periodic Reporting for period 4 - AutoRecon (Molecular mechanisms of autophagosome formation during selective autophagy)
Période du rapport: 2020-09-01 au 2021-02-28
Autophagy is an intracellular degradation system for the removal of harmful, bulky material from the cytoplasm. Autophagy thereby ensures cellular homeostasis and protects us from diseases such as neurodegeneration, cancer and uncontrolled infections. This is achieved by the sequestration of this material, referred to as the cargo, within double membrane organelles termed autophagosomes. Upon induction of autophagy, autophagosomes form de novo and are first observed as small membrane structures known as phagophores (or isolation membranes). These phagophores gradually expand and sequester the cargo as they grow. After closure of the phagophores, the resulting autophagosomes fuse with lysosomes (or the vacuole in yeast) wherein their inner membrane and the cargo are degraded. The biogenesis of autophagosomes is mediated by various proteins referred to as the autophagy machinery. How this machinery is orchestrated to generate autophagosomes was the subject of this project. Particular attention was given to selective autophagy, wherein defined cargo material is specifically targeted by the autophagy machinery. In order to obtain mechanistic insights into selective autophagy, especially with regard to which factors are sufficient for a given sub-reaction a reconstitution approach was conducted. These biochemical experiments were combined with cell and structural biology. Studying the cytoplasm-to-vacuole targeting pathway, a specialized selective autophagy-like process in yeast, as well as the selective autophagy of ubiquitinated proteins in mammalian cell, we found that the autophagy machinery is recruited to the cargo via cargo receptors. Thus, phagophores are not preformed but their formation is initiated directly at the cargo. Furthermore, we found that Atg9 vesicles, which are components of the autophagy machinery, function as platforms for the assembly of the autophagy machinery and thereby form seed for establishment of membrane contact sites driving the expansion of these vesicles into phagophores via lipid transfer.
Studying the cytoplasm-to-vacuole targeting (Cvt) pathway in yeast, we first found that the Atg12–Atg5-Atg16 complex, which is a key component of the machinery mediating the attachment of the Atg8 protein to the phagophore is recruited to the prApe1 cargo via the Atg19 cargo receptor. This suggested that cargo receptors don’t merely attach the cargo to Atg8 decorated membranes but that they might act upstream to generate these in the first place. We were further able to define the interaction site for Atg19 in Atg5 using computational approaches allowing us to assess the relevance of this interaction for the Cvt pathway in yeast. We then went on to include almost the entire autophagy machinery in reconstitution systems. The results obtained from them suggest that autophagy receptors mediate autophagosome formation directly at the cargo by recruiting Atg9 vesicles via scaffold proteins. The Atg9 vesicles in turn function as platforms for the assembly of the autophagy machinery, including the Atg2 protein. Atg2 establishes membrane contact sites with the endoplasmic reticulum, which in turn functions as donor compartment for the transfer of lipids into Atg9 vesicles for phagophore growth.
Our studies of mammalian selective autophagy revealed that cargo receptors are sufficient to sequester ubiquitinated proteins larger condensates. Subsequently, they recruit the autophagy machinery to these condensates via their interaction with the FIP200 scaffold protein, where this machinery mediates the attachment of Atg8 family proteins to the growing phagophore, which in turn are bound by the cargo receptors to sequester the cargo within autophagosomes. Our results were published in international journals and presented at international conferences.
Our studies of mammalian selective autophagy revealed that cargo receptors are sufficient to sequester ubiquitinated proteins larger condensates. Subsequently, they recruit the autophagy machinery to these condensates via their interaction with the FIP200 scaffold protein, where this machinery mediates the attachment of Atg8 family proteins to the growing phagophore, which in turn are bound by the cargo receptors to sequester the cargo within autophagosomes. Our results were published in international journals and presented at international conferences.
The insights into selective autophagy obtained within the framework of this project help to find ways to target the autophagy machinery to harmful material, accumulating within cells in pathological condition such as neurodegeneration.