LONDON’S GLOBAL UNIVERSITY Analysis of the novel Lipid transfer protein Anchored at Membrane contact sites (LAM) family A thesis submitted to the University College London in the fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Dr Tim Levine Department of Cell Biology, Faculty of Brain Science, Institute of Ophthalmology 2017 Declaration I, Louise Wong, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated. June 2017 1 2 Abstract Membrane contact sites are dynamic structures where two organelles come into close proximity to regulate and facilitate the flow of material and information between them. One type of inter-organelle communication is lipid exchange, which is essential for membrane maintenance and in response to environmental and cellular stimuli. We recently discovered a new family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) that is present in all eukaryotes. LAM proteins are integral Endoplasmic Reticulum (ER) proteins containing at least one domain that is structurally similar to the StARkin domain superfamily, a specialised fold that can bind amphipathic ligands such as lipids. The budding yeast, Saccharomyces cerevisiae, has six such proteins: Lam1p-6p. Lam1p-4p are located at contacts between the ER and the plasma membrane (PM), and Lam1p-3p are implicated in retrograde sterol traffic between the ER and PM. The PM contains a high concentration of sterol where it increases rigidity by altering the packing characteristics of the phospholipids in the bilayer. Sterol is also important in the ER, where its levels are low but it is both synthesised and sensed. However, the mechanism by which sterol traffics between the ER and the PM is unknown. This investigation characterises the phenotype of yeast delete LAM strains on Amphotericin B, a sterol sequestering antifungal agent and shows that the conserved StARkin domain of LAM proteins is responsible for resistance against Amphotericin B. Aspergillus fumigatus, a filamentous fungus, has two LAM proteins and the removal of AfLamA causes a severe growth phenotype. Also, in vitro studies indicate that LAM StARkin domains have a clear sterol transfer activity and a mutation that can diminish the function in vivo and in vitro has been identified. These findings present a new candidate protein family for intracellular sterol trafficking. 3 4 Dedication To my beloved brother, Howard Wong, 1985-2012 I dedicate this thesis to: my brother, you are dearly missed and always remembered and to my mother, for your encouragement and infinite support. 5 6 Acknowledgements I would like to thank my supervisor, Dr Tim Levine, for the guidance and inspiration through what has been an exciting and at times, a challenging project. Your expertise, creativity and passion have helped me become the scientist that I am and aspire to be for the rest of my career. I could not have learnt or achieved as much as I did without your supervision and influence and most significantly your tremendous enthusiasm for all things science! I am extremely thankful that you gave me the chance to work on such an original project and more importantly as part of your lab. The members of the Levine group have contributed immensely to my work and to my time in the lab: first and foremost, Alberto: thanks for being there from the start. It was my pleasure to have worked together on this project and also undertaking the PhD journey with you. I have no doubts that sharing lab and office space with me was not always easy with my ‘organised’ mess, so thanks for putting up with me! Sarah: your infectious energy inspired me when I needed motivation. Rachel: thanks for showing the way to becoming a fully-fledged PhD student. My time at the Institute of Ophthalmology has been made easier by many staff members who maintain equipment and support all of us here, thank you for your help. I am also deeply indebted for the expert discussions, advice and resources to all the brilliant scientists from all around the world whom I have met over the course of my PhD. I gratefully acknowledge the funding from the MRC that made my PhD possible. I also extend my gratitude to the Cockcroft lab for the lipid transfer expertise, Cordeiro lab (UCL) and Ben who has helped made many experiments possible and also the F2G Ltd lab (Manchester) who supported both my PhD funding and a significant part of this project especially Nicola. To all fellow lab/office members, past and present, I thank you for being there through the ups and downs of experiments and life, providing support and most importantly friendship (and also so much conversation that I thought I would never get any work done!). I am especially grateful to Ingrid and Tom for being late night office buddies – I’m sure I would have been more motivated to finish my work quicker if it were not for your excellent company. To all friends and family, I thank you for being part of my life. Now this thesis is done and I am no longer a student, I definitely do not have any excuse for not sharing the responsibility at the pub. To my family, a huge thanks for all their love and encouragement. To my parents, I am so grateful that you gave me the opportunities to study that you did not 7 have for yourselves and for your extraordinary support you have always offered. I hope that I have made you proud. To my brothers, you have been there throughout my life and, you influenced so much of who I am, thus I could not have started this journey without you. Finally, my dearest Thomas, you have been by my side throughout as my unwavering rock through the toughest and the best of times giving me unconditional support, care and friendship. The completion of my PhD has been a long journey and life did not stand still during this time. It is only due to your extraordinary patience, kindness and love that I am able to be here. You encouraged me from afar when I first started this PhD and then you gave me strength when my brother passed away. We have begun our life together in London, and I can never be happier than coming home to you (and our cats!). When life was the difficult during my diagnosis, surgery and recovery and also the long time before that when I was not quite right, you never left me alone. You supported me mentally, emotionally and physically and we prevailed because of you. Writing is not my forte, but you have tolerated my complaints and whining during the completion of this thesis. There are no words that can express my gratitude and appreciation for all that you have done and been for me. This achievement is as much yours as mine. I thank you with all my heart and soul for being you. 8 Contents Declaration ..................................................................................................................... 1 Abstract .......................................................................................................................... 3 Dedication ...................................................................................................................... 5 Acknowledgements ........................................................................................................ 7 List of Figures .............................................................................................................. 17 List of Tables ............................................................................................................... 21 List of Abbreviations .................................................................................................. 23 CHAPTER 1 Introduction ......................................................................................... 29 1.1 General Introduction .................................................................................... 29 1.2 Lipids and cellular membranes ................................................................... 29 1.2.1 Lipid bilayers ................................................................................................ 29 1.2.2 Function of sterol in a membrane ................................................................. 31 1.2.3 Regulation of cholesterol synthesis .............................................................. 34 1.2.4 Intracellular distribution of sterol ................................................................. 36 1.2.5 Sterol affinity in membranes ........................................................................ 37 1.2.6 Sterol pools ................................................................................................... 38 1.3 Intracellular lipid transfer ........................................................................... 38 1.3.1 Three classes of intracellular lipid movement .............................................. 38 1.3.2 Transport of cellular sterol between membranes .......................................... 39 1.3.3 Complications of unravelling intracellular sterol transport .......................... 40 1.3.4 Vesicular pathway is not responsible for bulk sterol transport .................... 41 1.3.5 Spontaneous diffusion is not sufficient ........................................................ 42 1.3.6 Lipid transfer by proteins at contact sites ..................................................... 43 1.4 Membrane Contact Sites .............................................................................. 44 1.4.1 ER membrane contact sites..........................................................................