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The Protein-Protein Interactome of Saccharomyces Cerevisiae ABC

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The Protein-Protein Interactome of Saccharomyces Cerevisiae ABC The Protein-Protein Interactome of Saccharomyces cerevisiae ABC Transporters Nft1p, Pdr10p, Pdr18p and Vmr1p by Asad Hanif A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Asad Hanif 2012 The Protein-Protein Interactome of Saccharomyces cerevisiae ABC Transporters Nft1p, Pdr10p, Pdr18p and Vmr1p Asad Hanif Master of Science Graduate Department of Molecular Genetics University of Toronto 2012 Abstract The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock. ii Acknowledgments I would like to thank my supervisor, Dr. Igor Stagljar for his help and support over the years. I would also like to thank my supervisory committee members, Dr. Leah Cowen and Dr. Christine Bear for their help and support towards my research project. I also want to thank Dr. Charlie Boone, Dr. Reinhart Reithmeier and Dr. Andy Fraser for being a part of my examination committee for my Master’s defense exam. I would also like to thank everyone in the Stagljar lab for their support. I would like to especially thank Dr. Jamie Snider for helping me with my project and research, and answering countless questions. I also want to thank Analyn Yu, Victoria Wong, Dr. Julia Petschnigg, Mehrab Ali and Dr. Mandy Lam for helping me with various aspects of my research project. I want to thank Mike Cox from Dr. Andrews’s lab for helping me with confocal microscopy. I want to thank Christoph Kurat from Dr. Andrews’s lab for helping me with qRT-PCR experiments and proving reagents for my experiments. I want to thank Bryan-Joseph San Luis from Dr. Boone’s lab for proving single and double deleted mutant strains for my experiments. I want to thank Simon Alfred from Dr. Nislow’s lab for helping me setup Tecan growth experiments. Finally, I want to thank my parents, brothers and family for their support. This was not possible without your encouragement. iii Table of Contents Abstract .......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iii List of Tables ................................................................................................................................ ix List of Figures ............................................................................................................................... xi List of Appendices ...................................................................................................................... xiii Abbreviations .............................................................................................................................. xiv Chapter 1: Introduction .................................................................................................................. 1 1.1: Research Objectives .......................................................................................................................... 2 1.2: ABC Proteins .................................................................................................................................... 2 1.2.1: Function of ABC Proteins .......................................................................................................... 2 1.2.2: Structure of ABC Transporters .................................................................................................. 4 1.2.3: ABC Transporters, Human Diseases and Multi-drug Resistance .............................................. 7 1.2.4: Human ABC Subfamilies ........................................................................................................... 9 1.3: ABC Transporters in S. cerevisiae .................................................................................................. 12 1.3.1: ABCC (MRP/CFTR) Subfamily in S. cerevisiae ..................................................................... 13 1.3.1.1: Nft1p ................................................................................................................................. 14 1.3.1.2: Vmr1p ............................................................................................................................... 15 1.3.2: ABCG (PDR5) Subfamily in S. cerevisiae .............................................................................. 18 1.3.2.1: Pdr10p ............................................................................................................................... 19 1.3.2.2: Pdr15p ............................................................................................................................... 23 1.3.2.3: Pdr18p ............................................................................................................................... 24 1.3.3: Other ABC Subfamilies in S. cerevisiae .................................................................................. 26 1.4: Protein-Protein Interactions, Model Organism, and Research Tools .............................................. 28 1.4.1: Importance of Protein-Protein Interactions .............................................................................. 28 1.4.2: S. cerevisiae as a Model System .............................................................................................. 29 1.4.3: Yeast Two-Hybrid (Y2H) ........................................................................................................ 31 1.4.4: Membrane Yeast Two-Hybrid (MYTH) .................................................................................. 34 1.4.5: Tandem Affinity Purification (TAP) and Mass Spectrometry ................................................. 37 1.5: Zinc Homeostasis in S. cerevisiae ................................................................................................... 38 1.5.1: Importance of Zinc ................................................................................................................... 39 1.5.2: Zap1p Transcription Factor ...................................................................................................... 40 1.5.3: Zinc Transporters ..................................................................................................................... 42 iv 1.6: Thesis Rationale .............................................................................................................................. 46 Chapter 2: Materials and Methods ............................................................................................... 48 2.1: General Experimental Protocols ..................................................................................................... 49 2.1.1: PCR Amplification ................................................................................................................... 49 2.1.2: DNA-Agarose Gel Electrophoresis .......................................................................................... 49 2.1.3: Standard Lithium Acetate Yeast Transformation ..................................................................... 50 2.1.4: DTT Method for Yeast Transformation ................................................................................... 51 2.1.5: Yeast Integration Transformation ............................................................................................ 51 2.1.6: Yeast and E. coli Miniprep ....................................................................................................... 52 2.1.7: Yeast and E. coli Miniprep in a 96-well Format ...................................................................... 53 2.1.8: Genomic DNA ......................................................................................................................... 53 2.1.9: Competent E. coli Preparation Using Inoue Method ............................................................... 53 2.1.10: E. coli Transformation ........................................................................................................... 54 2.1.11: Glycerol Stock Preparation for Yeast and E. coli .................................................................. 55 2.1.12: Sequencing DNA from TCAG/BioBasic ............................................................................... 55 2.1.13: FM4-64 Staining for Fluorescence Microscopy ..................................................................... 55 2.2: Bait Generation ..............................................................................................................................
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