Characterizing the assembly and molecular interactions of the fission yeast Atg1 autophagy regulatory complex by Tamiza Nanji B.Sc., M.Sc. (McMaster University) 2012, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2019 ©Tamiza Nanji, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Characterizing the assembly and molecular interactions of the fission yeast Atg1 autophagy regulatory complex submitted by Tamiza Nanji in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology Examining Committee: Calvin Yip, Biochemistry and Molecular Biology Supervisor Filip Van Petegem, Biochemistry and Molecular Biology Supervisory Committee Member Michel Roberge, Biochemistry and Molecular Biology Supervisory Committee Member Elizabeth Conibear, Medical Genetics University Examiner Chris Loewen, Cell and Developmental Biology University Examiner ii Abstract Macroautophagy, often referred to as autophagy, is a non-selective degradation mechanism used by eukaryotic cells to recycle cytoplasmic material and maintain homeostasis. Upregulated under starvation to generate molecular building blocks for ongoing cellular processes, this pathway requires the coordinated action of six multi-protein complexes, the Atg1/ULK1 complex being the first. Although, the Atg1 complex has been extensively studied in Saccharomyces cerevisiae, far less is known about the biochemical and structural properties of its mammalian counterpart, the ULK1 complex. Unlike the S. cerevisiae Atg1 complex which contains five subunits (Atg1, Atg13, Atg17, Atg29, and Atg31), the ULK1 complex consists of four proteins (ULK1, FIP200/RB1CC1, ATG13, and ATG101) that are technically more challenging to study. In this thesis, I characterized the Atg1 complex from fission yeast, Schizosaccharomyces pombe, as the composition of proteins resembles the mammalian ULK1 complex but is more amenable to biochemical analyses. The Atg1 complex in S. pombe is composed of Atg1 (ULK1 counterpart), Atg13, Atg17 (FIP200 counterpart) and Atg101. We found that the interactions between Atg1, Atg17, and Atg13 are conserved while Atg101 does not replace Atg29 and Atg31. Instead, Atg101 binds to Atg1 and the HORMA domain of Atg13. Furthermore, Atg101 was previously shown to contain a conserved loop, termed the WF finger, postulated to bind and recruit downstream autophagy-related proteins and effectors. Using affinity purification mass spectrometry, we further investigated the potential interacting partners of S. pombe Atg101 under autophagy-inducing and non-inducing conditions. We obtained 625 proteins that co-purified with Atg101-GFP from cells grown in defined media. We used in vitro pairwise studies to confirm the interaction between Atg101 and prey proteins. 9 of the 16 proteins tested were confirmed including Fkh1, an FKBP-type peptidyl-prolyl cis-trans isomerase. We further explored the interaction interface between Atg101 and Fkh1 and found that the WF finger is required for the interaction in vitro. Although the S. cerevisiae Fkh1 homologue, FKBP12, interacts with rapamycin; Fkh1 it is not thought to be directly involved in autophagy. Collectively, our results give new insights into iii an Atg101-containing Atg1/ULK1 complex and reveals that Atg101’s function may span beyond autophagy. iv Lay Summary All life forms consume nutrients and in turn produce waste. If not removed, waste accumulates leading to detrimental outcomes. Eukaryotic cells have evolved a mechanism termed autophagy (Greek for “self- eating”) to remove waste. Dysregulation in autophagy has been linked to devastating illnesses such as cancer. In this thesis, we explore the autophagy-related complex, Atg1, which is thought to help regulate autophagy initiation. Although the Atg1 complex has been extensively studied in budding yeast, the human Atg1 complex counterpart, the ULK1 complex, differs in subunit composition. We explored the Atg1 complex from fission yeast as it resembles the human ULK1 complex. We found that many of the interactions in the Atg1 complex are conserved between budding and fission yeast; however, the unique Atg101 protein forms interactions with Atg13HORMA and Atg1 within the Atg1 complex. We further explored potential Atg101 interactions. Our work suggests that Atg101’s function may extend beyond autophagy. v Preface This thesis contains contributions made by other scientists from around the world and includes a version of a published paper. These contributions are outlined below. A version of Chapter 2 was published as Tamiza Nanji, Xu Liu, Leon H. Chew, Franco K. Li, Maitree Biswas, ZhongQiu Yu, Shan Lu, Meng-Qiu Dong, Li-Lin Du, Daniel J. Klionsky & Calvin K. Yip (2017) Conserved and unique features of the fission yeast core Atg1 complex, Autophagy, 13:12, 2018-2027, DOI: 10.1080/15548627.2017.1382782. I took over this project from previous students, as such many of the constructs used for the pull down assays in Figure 2.1 were made by other students including Leon Chew, Katarina Priecelova, Tianlei Sun and Franco Li. I generated the remaining constructs needed and conducted all pull down assays and associated western blots to devise the S. pombe Atg1 complex interaction network (Figure 2.1, 2.2, 2.4). Electron microscopy (EM) studies of S. pombe Atg17 (Figure 2.3) were conducted by Leon Chew and Calvin Yip. Autophagy assays in the budding yeast were conducted by Xu Liu and Daniel J. Klionsky (University of Michigan). Autophagy assays in the fission yeast were conducted by ZhongQiu Yu and Li-Lin Du (National Institute of Biological Sciences, Beijing). I devised the study to compare the Atg1 complexes between fission and budding yeast with respect to Atg29 and Atg31. I generated the cells necessary and completed pull down assays and associated western blots for this section. With assistance from Dr. Calvin Yip, I purified the S. pombe Atg17-S. cerevisiae Atg31-Atg29 chimera complex and completed negative-stain EM analysis (Figure 2.5). Crosslinking experiments were completed by me, with limited assistance from Maitree Biswas (University of British Columbia). Shan Lu and Meng-Qiu Dong (National Institute of Biological Sciences, Beijing) analyzed our crosslinked samples using crosslinking mass spectrometry (CXMS) (Figure 2.6). Chapter 3 is an ongoing study. I generated the fission yeast Atg101-GFP construct in the S. pombe ARC039 strain and grew these cells for proteomic studies. Samples were prepared and analyzed using vi mass spectrometry by Aoki Hiroyuki and proteomic analysis was conducted by Sadhna Phanse from Dr. Mohan Babu’s group (University of Regina). Sadhna Phanse generated Figure 3.2C. I generated the constructs required for the in vitro pull down experiments to confirm interactions between Atg101 and our prey proteins. Atg101 mutants and subsequent cloning into expression vectors used to assess the interaction surface of Atg101 with Atg13HORMA and Fkh1 were prepared with the assistance of Diana Vasyliuk (Mitacs Globalink). I conducted the final pull down assays and associated western blots. vii Table of Contents Abstract ........................................................................................................................................................ iii Lay Summary ................................................................................................................................................. v Preface ......................................................................................................................................................... vi Table of Contents ....................................................................................................................................... viii List of Tables ................................................................................................................................................ xi List of Figures .............................................................................................................................................. xii List of Abbreviations and Symbols ..............................................................................................................xiii Acknowledgements ..................................................................................................................................... xvi CHAPTER 1: Introduction .............................................................................................................................. 1 1.1 Protein turnover helps preserve cellular homeostasis ........................................................................... 1 1.2 Two distinct mechanisms of cellular degradation: The ubiquitin-proteasome and lysosome- autophagy systems ....................................................................................................................................... 2 1.2.1 The ubiquitin-proteasome system (UPS) ............................................................................................. 2 1.2.2 The lysosome-autophagy system ......................................................................................................... 5 1.3 Historical milestones of lysosomal/vacuolar