Interaktion zwischen ATG2, ATG9 und WIPI4 und ihre Rolle in der Autophagosommembranbildung

Interaction between ATG2, ATG9, and WIPI4 and their role in autophagosome membrane formation.

by Pouya Hosnani
Doctoral thesis
Date of Examination:2025-02-07
Date of issue:2025-01-21
Advisor:Barbora Knotkova
Referee:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Michael Müller
Persistent Address: http://dx.doi.org/10.53846/goediss-11017

 

 

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Abstract

English

Autophagy is a cellular recycling mechanism involved in various physiological functions, such as homeostasis, cellular stress response, and nutrient deprivation. A key step in autophagy is the formation of the autophagosome membrane. Autophagy-related gene protein (ATG)2 is a lipid transfer protein that plays a crucial role in membrane formation by supplying lipids. In this process, ATG2 interacts with other proteins, such as WD-repeat protein Interacting with PhosphoInositides (WIPI)4 and ATG9. WIPI4 acts as a membrane anchor for ATG2 and helps bind the C-terminal end of ATG2 to the membrane. No function of WIPI4 independent of protein-protein interaction is currently known. ATG9 is the only known transmembrane protein involved in the autophagy process. In recent years, it has been demonstrated that ATG9 possesses a lipid-scrambling function. Additionally, a direct interaction between ATG9 and ATG2 has been identified. It is therefore hypothesized that ATG9 may accelerate lipid transfer by ATG2. ATG9 is believed to balance membrane asymmetry between the outer and inner membrane layers, which arises from lipid transfer to the outer membrane layer. Using the giant unilamellar vesicle (GUV) assay, I was able to demonstrate the protein binding of WIPI4 and ATG2 to membranes and the phosphatidylinositol 3-phosphate (PI3P)-dependence of the process. Furthermore, I showed ATG2-induced clustering of lipid vesicles. I observed that ATG2 accumulates in a ring-like structure at the contact site between two membranes and draws the membranes closer together. Additionally, I microscopically demonstrated the interaction of ATG9-containing large unilamellar vesicles (LUVs) with PI3P-containing GUVs in the presence of ATG2. With this, I have laid the foundation for further investigations in this field, aiming to visually represent autophagosomal membrane formation. To study how ATG9 promotes autophagosome formation, I measured the effect of ATG9 on lipid transfer by ATG2 in a lipid transfer assay. I found that ATG9 increases the lipid transfer rate by a factor of 2.38. This confirms the important role of ATG9 in membrane expansion of the autophagosome. The exact mechanism by which ATG9 accelerates lipid transfer by ATG2 remains unclear. Possible factors include direct interaction, lipid-scrambling activity, and membrane-bending properties of ATG9. To enhance the effect of ATG9, additional ATG proteins such as WIPI4 and ATG13/101 can be included. To better understand the process of autophagosome formation, further ATG proteins can be incorporated into the lipid transfer assay in the future.
Keywords: autophagy; atg2; atg9; wipi4; biochemie