Architecture and function of the phagosome assembly site in autophagy

Autophagy is an intracellular degradation pathway by which cytosolic components and organelles are sequestered into a double-membrane vesicle and then delivered to the vacuole/lysosome for breakdown and recycling. It mediates cell homeostasis by the degradation of long-lived proteins and entire organelles. Autophagy also contributes to the adaptive response to starvation and various other stresses. Not surprisingly, defects in autophagy have been associated with several human diseases, including neurodegenerative disorders and cancer. During autophagy, membranes are initiated at the phagosome assembly site (PAS) close to the vacuole. The formation of these organelles called autophagosomes is regulated by the Atg1 kinase complex present at this location. Several components essential for autophagy induction have been identified and many of them are conserved from yeast to mammals, yet the mechanisms underlying this event remain elusive. As autophagosomes are not constitutively present in a cell but only formed upon certain stimuli, studying the formation of these organelles also helps us to understand organelle biogenesis in general. Knowledge of the PAS components and their interactions and dynamics is key to understanding how autophagy is regulated. I will dissect the architecture and function of the PAS and analyze the role of the Atg1 kinase complex in building this structure. I will use budding yeast as the main model system and apply a unique combination of biochemical and cell biological approaches in vitro and in vivo. These approaches will greatly advance our understanding of autophagy regulation and address a number of key questions on the regulation of organelle formation in general. Since many factors are evolutionary conserved in higher eukaryotes, this work will also help us to better understand diseases associated with autophagy misregulation.

A clean apartment and workplace, while certainly important, are not strictly necessary in order to survive. For cells, however, tidying up is absolutely vital. The responsible process is called autophagy, which has now become widely known due to Yoshinori Ohsumis winning of the Nobel Prize in Medicine in October 2016. During autophagy, a defined set of proteins coordinates the removal of viruses, bacteria, and damaged or superfluous material from a cell. Autophagy also enables cells to survive times of starvation, by degrading the cells own components to recycle their building blocks similar to recycling stations in a town. This process needs to be tightly controlled to prevent the removal of structures that are still required in the cell. For some time, researcher had known the key coordinator of autophagy the protein Atg1. However, how Atg1 exactly does its job remained unclear. In this project, we showed that Atg1 modifies a set of proteins with a specific recognition sequence. We not only deciphered the recognition sequence but also determined the cellular proteins that actually contain this sequence. One, Atg9, caught our interest as it is not only a known component of the cellular 'waste bag', but also contains a stunning six of those specific recognition sequences. Every cell contains specialized recycling stations. In order to dispose its waste, a cell needs to wrap up the waste, similar to putting garbage in a bag. This cellular trash bag can then be brought to the cellular recycling station where the waste is broken down to re-usable parts. Atg9 is essential for this process. We also identified a new player in autophagy, the protein Hrr25, which coordinates the selection of waste. Furthermore, we investigated how Atg1 and the process of autophagy are controlled in order to prevent their aberrant activation. In a normal cell two coordinators bring Atg1 and the waste independently from each other to the waste packaging location. When these coordinators are removed from the cell, the waste and Atg1 cannot meet and autophagy is not induced. In cells without these coordinators, we were able to promote waste removal by autophagy when Atg1 was artificially forced to meet the waste. This shows that the concurrence of Atg1 and waste at the right place is a key regulatory step to activate autophagy. The detailed study of such fundamental cellular processes is crucial for the understanding of diseases that go hand in hand with these events in the case of autophagy, Alzheimers disease or cancer. In the long run, this will help to better treat or perhaps even prevent these illnesses.