University of Groningen
New insights into autophagy regulation using yeast Saccharomyces cerevisiae Rodrigues de Abreu, Susana
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New insights into autophagy regulation
using yeast Saccharomyces cerevisiae
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Colophon
Cover: “A journey to the center of a starving budding yeast” Cover Design: Alessia Peviani || www.photogenicgreen.com Layout: Susana de Abreu
New insights into autophagy regulation using yeast
Saccharomyces cerevisiae
PhD thesis, University of Groningen, The Netherlands
©Susana de Abreu , Groningen 2018
ISBN/EAN: 978–94–034–1197-2
Printed by: ProefschriftMaken || www.proefschriftmaken.nl
All rights reserved. No part of this publication may be reproduced, distributed or transmitted in any form or by any means without prior permission by the author. The copy right of the articles that have been published or have been accepted for publication has been transferred to the respective journals. The printing of this thesis was financially supported by the University of Groningen, the University Medical Center Groningen and Graduate School of Medical Sciences.
New insights into autophagy regulation
using yeast Saccharomyces cerevisiae
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of
the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 10 December 2018 at 12:45 hours
by
Susana Rodrigues de Abreu
born on 12 January 1983 in Viana do Castelo, Portugal
IV Supervisors Prof. F. Reggiori Prof. D. Hoekstra Assessment committee Prof. J. Klumperman Prof. S. van Ijzendoorn Prof. K. Thedieck
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Table of Contents
Aim and scope of the thesis ... 7 Chapter 1:
General Introduction ... 13
Chapter 2:
The origin of autophagosomes: the beginning of an end ... 57
Chapter 3:
Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation ... 79
Chapter 4:
Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation 121
Chapter 5:
Screening for components of the Atg machinery involved in Atg9 trafficking ... 159
Chapter 6:
Lipid partitioning at the nuclear envelope controls membrane biogenesis ... 187
Chapter 7:
A neurotoxic glycerophosphocholine impacts PtdIns-4,5-bisphosphate and TORC2 signaling by altering ceramide biosynthesis in yeast ... 229
Chapter 8:
General Discussion ... 271
Chapter 9:
Summary and Nederlandse Samenvatting ... 287
Chapter 10:
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Aim and scope of the thesis
Autophagy is a major cellular catabolic process essential to maintain cell homeostasis. It participates in the elimination of excess or damaged cell components and invading pathogens, but also in the recycling of cellular content during nutrient deprivation. Depending on the targeted material selected to be degraded, autophagy can be either a non-selective or a non-selective process. The induction of autophagy leads to the formation of an initial cistern, the phagophore, at a site known as the phagophore assembly site (PAS), which expands sequestering the cellular content. When the phagophore seals, a double membrane vesicle termed the autophagosome, is generated. Upon the fusion of the outer membrane of this large vesicle with the vacuole/lysosome, the cargo is released into the lumen of this organelle where it is degraded by hydrolytic enzymes. The resulting metabolites are reused either for synthesis of new macromolecules or as an energy source. The molecular machinery mediating the formation of autophagosomes is composed by the so-called autophagy-related (ATG) proteins, which have been identified mostly with genetic screens in yeast. In the last two decades, a continuously growing number of studies has led to many advances on our knowledge about autophagy, the origin of the autophagosomal membranes, the mechanism of autophagosome formation and fusion, the signalling cascades regulating autophagy and the physiological roles of this pathway. As in many other fields, however, multiple aspects of autophagy are awaiting to be completely clarified. Autophagy plays an important role in numerous human disorders, mainly functioning as a protective mechanism, and therefore there is an enormous interest in exhaustively understanding this process in order to be able to manipulate it for the benefit of the human health. The goal of this thesis is to contribute with new knowledge that integrated with the previous or future studies, will help to better decipher the autophagy process, specifically its regulation.
I begin this thesis by providing an overview on our current knowledge about the autophagy, covering diverse aspects of this pathway. In particular, I describe the overall mechanism of autophagy, why yeast has been a key model for the study of autophagy, the molecular
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machinery involved in this process, the main regulatory signalling pathways and some of the physiological functions of autophagy (Chapter 1).
The autophagosome is not originated through budding from a pre-existing organelle, but it appears to be formed de novo. The origin of the lipid bilayers that constitute the autophagosome, however, is still one of the main enigmas in the field of autophagy. In Chapter 2 we provide an overview on this topic, based on the results obtained by different studies, which highlight possible autophagosome membrane sources.
Atg4 is a cysteine protease that cleaves Atg8 post-transcriptionally. This primed version of Atg8 subsequently gets conjugated with the phosphatidylethanolamine (PE) present in the membranes at the PAS upon autophagy induction. There, Atg8 plays a crucial role in the autophagosome formation. Atg4 is also responsible for the deconjugation of Atg8-PE and consequent release of Atg8 from the surface of complete autophagosomes. The main aim of this thesis was to investigate how autophagy process is regulated specifically by Atg4 activity through Atg8-PE deconjugation from the autophagosome (Chapters 3 and 4). In Chapter 3, we explore the molecular mechanisms mediating Atg4 recruitment to the PAS, which we show to be essential for its Atg8-PE deconjugation activity. We also demonstrate that Atg4 association to the PAS is mediated by its direct binding to Atg8-PE through a domain that we called APEAR. In
Chapter 4, we identified an upstream factor that regulates Atg4 activity
and prevents Atg8-PE cleavage. We found that Atg1 phosphorylates Atg4 S307, a modification that inhibits its catalytic activity during the autophagosome formation and impedes a constitutive removal of Atg8 from autophagosomal membranes. The findings described in Chapters
3 and 4 were essential to understand Atg4 regulation mechanisms, and
how this regulation plays a crucial role in the autophagosome biogenesis.
Atg9, another Atg protein essential for autophagy, was shown to shuttle between its cytoplasmic clusters of membranes and the PAS. Some factors have been linked to this Atg9 trafficking, but the role of all the
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components of the core Atg proteins was not systematically assessed yet. In Chapter 5, we performed a fluorescence microscopy-based screen in yeast where we analysed the distribution of Atg9-GFP in absence of each one of the core Atg proteins under selective or non-selective autophagy conditions. We discovered that additional Atg proteins participate in the trafficking of Atg9, most of them under a specific type of autophagy. Our results open the door to new and promising investigations, to better understand the roles of Atg9 in autophagy.
Autophagy is part of a complex and integrated response of the cells to stress conditions such as nutrient deprivation. When nutrients are scarce, cells optimize their energy consumption by reorganizing their metabolism and their energy storage. In Chapter 6, in collaboration with Siniossoglou group, we investigated the mechanism involved in the switch from phospholipids and membrane biogenesis, to lipid storage in lipid droplets (LDs) under nutrient depletion conditions. We revealed that the yeast phosphatase Pah1 plays a central role in this process. Nutrient-depletion induces Pah1 translocation to the nuclear membranes where it is involved in channelling cell lipids from phospholipid biosynthesis and membrane biogenesis to their storage into LDs.
Autophagy and LDs have been tightly associated, LDs have been shown to be degraded via autophagy through lipophagy and LDs have also been implicated in the autophagosome biogenesis. Therefore, a more in-depth knowledge about LDs biogenesis could potentially contribute to a better understanding of autophagosome biogenesis itself. Lipid homeostasis appears to be compromised in several pathologies including Alzheimer’s disease (AD). In AD patients, the specific phosphatidylcholine species belonging to platelet activating factor family, PC(O-16:0/2:0), is particularly elevated. The increased levels of this lipid have been shown to disturb the cell lipid metabolism with toxic consequences for neurons. In Chapter 7, we participated to a study of the Baetz group, where we investigated the signalling pathways that mediate the toxic effects of PC(O-16:0/2:0) in yeast, to gain insights into what happens in neurons. We found that elevated levels of
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PC(O-16:0/2:0) promote accumulation of PtdIns(4,5)P2 at the plasma
membrane, which in turn cause an inhibition of TORC2 signalling. We further showed that the effect of PC(O-16:0/2:0) on PtdIns(4,5)P2
distribution are in part linked to changes in long chain bases and ceramides biosynthesis. These lipid and signalling alterations may explain the toxicity associated with AD and become potential therapeutic targets to modulate AD pathology progression.
In Chapter 8 I summarize the most relevant outcomes of this thesis and discuss future research perspectives.
