Open Research Online The Open University’s repository of research publications and other research outputs Characterisation of human H-ATPase a4 subunit Thesis How to cite: Su, Ya (2004). Characterisation of human H-ATPase a4 subunit. PhD thesis The Open University. For guidance on citations see FAQs. c 2004 Ya Su Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000f9f2 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk CHARACTERISATION OF THE HUMAN H^ATPase a4 SUBUNIT by Ya Su B.Sc. (Hons.), M.Sc. Submitted to the Open University (Life Sciences) for the Degree of Doctor of Philosophy- August 2004 Addenbrooke’s NHS Trust, Department of Medical Genetics Hills Road, Cambridge. University of Cambridge U.K. U.K. ProQuest Number: 27527259 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 27527259 Published by ProQuest LLO (2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT The vacuolar H^-ATPase (or V-ATPases) are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialised sites such as kidney epithelial cells and osteoclasts, as well as acidifying intracellular compartments. The 100 kDa polytopic a subunit of this group of ATPases is suggested to play important roles in proton translocation, assembly, and targeting as well as coupling of ATP hydrolysis and proton transport of the V-ATPase. In man, different a subunit paralogues are encoded by four genes. ATP6V0A4 encodes a4, which is dominantly expressed apically in «-intercalated cells in both human and mouse kidney. I sought binding partners for a4 in order to address its potential role in the V-ATPase complex. Random peptide phage display analysis using a4's C terminus as a target protein revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by phage ELISA. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from human kidney membrane proteins. An in vitro PFK-1 pull-down assay showed that this interaction is also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co- immunolocalised with a4 in a-IC in the collecting ducts of human kidney. These findings indicate a direct link between V-ATPase and glycolysis, via the C-terminus of the pump's a subunit, and suggest a novel regulatory mechanism between V-ATPase function and energy supply. This interaction between the a subunit and PFK-1 also provides new evidence that the C-terminus of this subunit lies cytoplasmically in vivo. Finally, SPR analysis suggests a possible alteration of the a4/PFK-1 interaction by the mutation (G820R) within the a4(G) region identified from a patient with rdRTA, providing a potential mechanism for disease. TO MY PARENTS, HUSBAND, AND DAUGHTER FOR THEIR ASSISTANCE, ENCOURAGEMENT AND LOVE. DECLARATION I declare that the work contained in this thesis, submitted by me for the degree of Doctor of Philosophy, is my own original work, except where explicit reference is made to the work of other authors. It has not been previously submitted for a higher degree or any other qualification. Signed Date TABLE OF CONTENTS ACKNOWLEDGEMENTS................................ v RELATED PUBLICATIONS............................................................................................vi ABBREVIATIONS.........................................................................................................vil CHAPTER 1: INTRODUCTION............................................... 1 1.1 The Vacuolar H'^-ATPase (or V-ATPase) ........................ 1 1.2 Interactions Involving Subunits of the V-ATPase ................................. ........... 21 1.3 The a Subunit of the V-ATPase .................. 24 1.4 The Aims of the Present Study ................... 44 CHAPTER 2: MATERIALS AND METHODS.................... 45 2.1 Chemicals, Enzymes and Antibodies ...............................................................45 2.2 General Buffers, Bacterial Growth Media and Antibiotic Solutions ................... 45 2.3 Cell Strains and Storage ..................................................................................45 2.4 Plasmids .................................................................................................45 2.5 DNA Analysis .................................... 46 2.6 Purification of DNA ........................... 47 2.7 Design, Synthesis and Storage of Oligonucleotides (Primers) ..........................48 2.8 Polymerase Chain Reaction (PGR) Methods ................................................... 48 2.9 Site-Directed Mutagenesis ...............................................................................50 2.10 Gene Cloning ...................................................................................... 52 2.11 DNA Sequencing ................................. 54 2.12 Protein Quantification and Detection .......... 57 2.13 Protein Expression ....................................................... 62 2.14 Protein Purification .............................................. 6 6 2.15 Protein Refolding .............................................................................................69 2.16 Human Kidney Protein Fractionation ................................................................ 70 2.17 Circular Dichroism (CD) Spectroscopy ............................................................. 71 2.18 N-terminal Sequence Analysis ............... 71 2.19 Identification of Protein Ligands........................................................................72 2.20 Protein Labelling: Biotinylation of Protein ..........................................................80 2.21 Characterisation of Protein-Protein Interactions ................................................80 2.22 Binding Affinity Study by Surface Plasmon Resonance (SPR) ..........................82 2.23 Immunohistochemistry ..................................................................................... 83 CHAPTER 3: EXPRESSION AND PURIFICATION OF a4(N), a4(Loop2) AND a4(C)...84 3.1 Introduction ...................................................................................................... 84 3.2 Results ............................................................................................................ 8 6 3.3 Discussion ..................................................................................................... 117 CHAPTER 4: SCREENING FOR BINDING PARTNERS..............................................124 4.1 Introduction ................................... 124 4.2 Results .......................................................................................................... 128 4.3 Discussion .......................................... 150 CHAPTER 5: CHARACTERISATION OF INTERACTION BETWEEN a4(C) AND ITS POTENTIAL BINDING PARTNER PFK-1.....................................................................155 5.1 Introduction .................................................................................................... 155 5.2 Results .......................................................................................................... 157 5.3 Discussion ................................................................................. 174 CHAPTER 6: CONCLUSION AND GENERAL DISCUSSION......................................179 6.1 Conclusion ..................................................................................................... 179 6.2 General Discussion .........................................................................................181 APPENDIX .............................................................................................................193 A.1 General Buffers, Bacterial Growth Media and Antibiotic Solutions ...................... 193 A.2 Antibodies, Cell Strains, Plasmids and Primers ..................................................194 REFERENCE...............................................................................................................202 LIST OF FIGURES Figure 1 Schematic models of V-ATPase and F-ATPase .................................................11 Figure 2 A Schematic 2D diagram of the human a4 subunit (not to scale). ......................26 Figure 3 Structure of a nephron ...................................................................................... 36 Figure 4 a- and p-intercalated cell ...................................................................................40 Figure 5 SDS-PAGE analysis ............................. 89 Figure 6 SDS-PAGE analysis .................................................................... 90 Figure 7 SDS-PAGE