This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Caspase interaction of anti‑apoptotic Livin as well as the Vacuolar ATPase (V‑ATPase) and structural insights into the subunit d and a of the yeast V‑ATPase Thaker, Youg Raj 2009 Thaker, Y. R. (2009). Caspase interaction of anti‑apoptotic Livin as well as the vacuolar ATPase (V‑ATPase) and structural insights into the subunit d and a of the yeast V‑ATPase. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/19015 https://doi.org/10.32657/10356/19015 Downloaded on 25 Sep 2021 15:51:45 SGT CASPASE INTERACTION OF ANTI-APOPTOTIC LIVIN AS WELL AS THE VACUOLAR ATPASE (V-ATPASE) AND STRUCTURAL INSIGHTS INTO THE SUBUNIT d AND a OF THE YEAST V-ATPASE YOUG RAJ THAKER SCHOOL OF BIOLOGICAL SCIENCE 2009 Caspase interaction of anti-apoptotic Livin as well as the vacuolar ATPase (V-ATPase) and structural insights into the subunit d and a of the yeast V-ATPase YOUG RAJ THAKER School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2009 Acknowledgments i Acknowledgements My deepest and sincere gratitude goes to my supervisor A/Prof. (Dr.) Gerhard Grüber for his continuous guidance, scientific advice, making in me a strict discipline of science, and taking care of myself and my scientific interests when I needed it most. Without his generous support, kind heartedness and devotion to the science, I wouldn’t have been able to complete this thesis. Many thank to A/Prof. Gerhard Grüber for giving me an opportunity to work under his guidance, and adopting me as a part of his group at a very critical time. This has been a great pleasure to work under the guidance of A/Prof. Gerhard Grüber. His attitude towards the life and science gave me an unforgettable experience, which will surely help me to succeed in the science and research. I would also like to take this opportunity to thank A/Prof. Wu Mian for his initial guidance at School of Biological Science and helping me to initiate a research interest in apoptosis. Special thanks go to A/Prof. Gerhard Grüber and A/Prof. Manfred Roessle for their help in collecting the Small Angle X-ray (SAXS) Data of the subunit d at EMBL, Hamburg Outstation (Germany). Appreciate help from A/Prof. Stephan Wilkens (Upstate Medical University, New York, USA) for providing high quality images of 3D electron micrographs of the VO domain of clathrin coated vesicles and to Dr. Ardina Grüber for help and discussion with MALDI-TOF data analysis. I would like to extend my thanks to A/Prof. Thorsten Wohland for collaboration about subunits a and d FCS binding studies with the support of Dr. Pan Xiaotao from his lab. I am grateful to colleague Mr. Shovanlal Gayen for help with NMR spectroscopy and effective discussions and to all lab members for creating a lively environment and moral support. Thanks go to Dr. Cornelia Hunke for help with computer related problems. Again, I very much appreciate A/Prof. Gerhard Grüber for critical comments and reading this thesis several times. Thanks also go to everyone who helped in thesis reading and corrections. Special thanks go to the Nanyang Technological University for providing me an opportunity to study in Singapore and financially supporting me. I am extremely grateful to my girl friend, Manisha Mishra for her immense support and care in my life. Last but not least, my heartedly gratitude goes to my parents for selfless support, love and understanding during and before my PhD study in Singapore. I would also like to remember my grandfather for his kind heartedness, love and best wishes I am living with, who passed away during my PhD study at NTU (Singapore). ii To my grandparents, parents and to all those who love me and care about me! Contents iii Acknowledgements .................................................................................................................... i Contents .............................................................................................................................. ......iii List of figures ............................................................................................................................ vi List of tables ............................................................................................................................ vii Abbreviations ......................................................................................................................... viii Abstract ..................................................................................................................................... x 1. INTRODUCTION…………………………………….…………………………………...1 1.1 Apoptosis and V-ATPase ..................................................................................................................... 2 1.1.1 Overview of apoptosis ......................................................................................................................... 2 1.1.2 ARTS role in the programmed cell death .............................................................................................. 6 1.1.3 Role of anti-apoptotic IAPs in apoptosis ............................................................................................... 8 1.1.4 Livin function in apoptosis ................................................................................................................... 8 1.2 Eukaryotic V1VO ATPase (V-ATPase) ............................................................................................... 11 1.2.1 Function and regulation of V1VO ATPase ........................................................................................... 11 1.2.2 Structural feature of V1VO ATPase ..................................................................................................... 18 1.2.3 Subunit d of V-ATPase ...................................................................................................................... 19 1.2.4 Subunit a of V-ATPase ...................................................................................................................... 22 1.3 Goals of this thesis ............................................................................................................................. 28 2. MATERIALS AND METHODS………………………………………………………...30 2.1 Materials ........................................................................................................................................... 31 2.1.1 Chemicals.......................................................................................................................................... 31 2.1.2 Molecular biology materials ............................................................................................................... 31 2.1.2.1 Templates, primers and peptides ........................................................................................................ 31 2.1.2.2 Enzymes and kits .............................................................................................................................. 31 2.1.2.3 Cell Lines, Yeast and E. coli strains .................................................................................................. 32 2.1.2.4 Vectors ............................................................................................................................................. 32 2.1.2.5 Antibodies ......................................................................................................................................... 32 2.1.2.6 Caspase inhibitors .............................................................................................................................. 32 2.1.3 Chromatography ................................................................................................................................ 33 2.1.3.1 Ion exchange ..................................................................................................................................... 33 2.1.3.2 Gel filtration ...................................................................................................................................... 33 2.1.3.3 Instruments and accessories ............................................................................................................... 33 2.1.3.4 Protein concentration, estimation and dialysis .................................................................................... 33 2.1.4 Other instrumentation ........................................................................................................................ 33 2.1.5 Softwares .......................................................................................................................................... 34 2.2 Methods ............................................................................................................................................ 37 2.2.1 Yeast genomic DNA isolation ............................................................................................................ 37 2.2.2 PCR cloning and mutagenesis of ARTS, Livin, subunit d and subunit a constructs .............................. 37 2.2.2.1 Vector preparation ............................................................................................................................. 37 2.2.2.2 Cloning and mutagenesis of Livin and ARTS constructs ....................................................................