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Characterization of Interactions in the Tap Family of Half

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Characterization of Interactions in the Tap Family of Half CHARACTERIZATION OF INTERACTIONS IN THE TAP FAMILY OF HALF ABC TRANSPORTERS. By Dennis Brian Leveson-Gower B.Sc, The University of Victoria, 1999. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology) The University of British Columbia April 2005 © Dennis Brian Leveson-Gower, 2005 ABSTRACT The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) protein which transports peptides for presentation to the immune system. TAP is composed of two half transporters, TAP1 (ABCB2) and TAP2 (ABCB3) which require heterodimerization for function. In humans, the TAP gene family consists of TAP 1, TAP2, and TAPL (ABCB9). While the TAP1-TAP2 complex is well-characterized, the dimerization state and function of TAPL are unknown. To identify interactions within the human TAP family, I adapted the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to study the human genes coding for the half ABC transporters. This assay has been used for the study of membrane-bound proteins in vivo (Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Science 283(5404), 990-3). With this method, in vivo TAP1-TAP2 heterodimerization was confirmed, no homodimerizations were detected with TAP1 or TAP2, and TAPL did not show any interaction with TAP1 or TAP2. However, I found strong evidence that TAPL forms homodimers. These results provide evidence of a novel homomeric TAPL interaction and demonstrate that the DHFR PCA will be of general utility in studies of half ABC transporter interactions in vivo. By using an insect-cell microsomal transport assay, I found the first direct evidence that TAPL can transport peptides. Two classical TAP substrates, RRYQNSTEL and RYWANATRST were transported by TAPL. Kinetic characterization of the transport of RYWANATRST indicated that TAPL follows Michaelis-Menton kinetics with an apparent Km of 3.2 ± 0.4 nM. TABLE OF CONTENTS Abstract ii Table of contents iii List of tables v List of figures vi List of Abbreviations viii Acknowledgements x I Introduction 1 1.1 — ABC transporters 1 1.2 — Half ABC transporters: a need for dimerization 6 1.3 — Methods for detecting interactions between half ABC trasnporters 7 1.4 — Thesis objectives 9 Bibliography 11 II Application of the DHFR PC A to TAP family of half ABC Transporters 17 2.1 - Introduction: A need to assay for interactions in the TAP family 17 2.2 - Materials and Methods 21 2.2.1 — Creation of DHFR PCA vectors 21 2.2.2 — Cell lines and culture 29 2.2.3 — DHFR survival assay 29 2.2.4 —FACS 30 2.3 - Results 31 2.3.1 — DHFR PCA survival-selection in nucleotide-free media 31 2.3.2 — FACS analysis of clones 32 2.4 - Discussion 35 Bibliography 37 III Utility of the DHFR PCA for the study of ABC Transporters 42 3.1 - Introduction 42 3.2 - Material and methods 46 3.2.1 — Cell lines and culture 46 3.2.2 — Western blots 46 3.2.3 — RNA purification and reverse transcription 47 3.2.4 — PCR of false positive transcripts 48 3.2.5 — MHC Class I expression assay 48 3.2.6 —FACS 49 3.3-Results 50 3.3.1 — Western blot analysis of protein expression in colonies 50 3.3.2 — Characterization of transcripts expressed in false positive colonies 51 3.3.3 — Effect of DHFR fusion on TAP1 and TAP2 function 53 3.4 - Discussion 56 Bibliography 58 IV Peptide transport by TAPL 59 4.1 - Introduction 59 4.1.1— Peptide transport by TAP 1/2 59 4.1.2 — Why TAPL may be a peptide transporter 64 4.2 - Materials and Methods 68 4.2.1 — Creation of a TAPL expression vector for insect cells 68 4.2.2 — Maintenance, transfection, and selection of insect cells 68 4.2.3 — Western blots 69 4.2.4 — Microsome preparation 70 4.2.5 — Peptide transport assay 71 4.3 - Results 74 4.3.1 — Creation of a TAPL-expressing cell line and preparation of microsomes 74 4.3.2 — Peptide transport assay 75 4.4 - Discussion 81 Bibliography 83 V Overall Discussion and Future prospects 89 5.1 - Discussion 89 5.2 - Future prospects 94 5.2.1 — Peptide specificity of TAPL 94 5.2.2 — Localization of TAPL homodimers 95 5.2.3 — Function in immune tolerance 96 5.2.4 — Detection of interactions within the White family of half ABC transporters... 96 Bibliography 98 LIST OF TABLES Table 2.2.1: Primers used for the sub-cloning of TAP family cDNAs into DHFR PCA vectors LIST OF FIGURES Figure 1.1.1: X-ray crystallography structure of Escherichia coli MsbA 3 Figure 1.1.2: Potential model for lipid A transport by Escherichia coli MsbA 5 Figure 2.2.1: Crystal structure of DHFR 22 Figure 2.2.2: Sub-cloning of zip-DHFR positive controls 23 Figure 2.2.3: Creation of parental DHFR vectors with 10 amino acid linkers 24 Figure 2.2.4: Modifying DHFR parental vectors to contain 20 aa linkers 25 Figure 2.2.5: Maps of DHFR constructs 28 Figure 2.3.1: Colony counts from survival assay 32 Figure 2.3.2: Relative increase in fluorescence between non-transfected cells and colonies from survival-selection 34 Figure 3.1.1: Possible origins of false positive colonies 43 Figure 3.1.2: MHC class I expression pathway 45 Figure 3.3.1: Western blot analysis of clones from the DHFR survival assay 51 Figure 3.3.2: RT-PCR detection of transcripts expressed in colonies from the survival assay 53 Figure 3.3.3: FACS of MHC class I expression 55 Figure 4.1.1: Schematic model of TAP 61 Figure 4.1.2: Substrate recognition motif and substrate binding pocket of human TAP 63 Figure 4.1.3: Homology between peptide binding regions of TAP1, TAP2, and TAPL 65 Figure 4.1.4: A working model for phagosomal cross-presentation 66 Figure 4.2.1: Determining direction of peptide transport 73 Figure 4.3.1: Selection of TAPL-His expressing High Five cells 74 Figure 4.3.2: ATP-dependent transport of RRYQNSTEL by TAPL 76 Figure 4.3.3: Peptide transport assay with peptides FAPGNYPAL and RYWAN ATRST 77 Figure 4.3.4: Transport of radioactive 0.39 nM RYWANATRST as a function of time 79 Figure 4.3.5: Lineweaver-Burk plot of initial velocity as a function of substrate concentration for TAPL's transport of RYWANATRST by TAPL 80 Figure 5.1.1: Proposed functions of TAPL 93 LIST OF ABBREVIATIONS ABCB2 ATP-binding cassette sub-family B transporter 2 ABCB3 ATP-binding cassette sub-family B transporter 3 ABCB9 ATP-binding cassette sub-family B transporter 9 bp base pair(s) CFTR Cystic fibrosis transmembrane conductance regulator CHO Chinese Hamster ovary CMV cyto-megalo virus DHFR PCA dihydrofolate reductase protein-fragment complementation assay ER endoplasmic reticulum FACS fluorescence-activated cell sorting FBS Fetal Bovine Serum FRET fluorescence resonance energy transfer HT hypoxanthine and thymidine MHC Major Histocompatibility Complex NBD Nucleotide binding domain Ova Chicken egg ovalbumin PBS phosphate buffered saline PCR polymerase chain reaction RE restriction endonuclease RT reverse transcription TAP transporter associated with antigen processing TAP1 transporter associated with antigen processing 1 also known as ABCB2 TAP2 transporter associated with antigen processing 2 also known as ABCB3 TAPL TAP-Like protein also known as ABCB9 TMD Transmembrane domain TM Transmembrane helix SDS Sodium Dodecyl Sulfate UTR untranslated region VSV vesicular stomatitis virus. ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Victor Ling, for giving me the opportunity to work in his lab and for constantly challenging me. He taught me how to think for myself and has given me a great foundation for doing science. It was a great pleasure to work with the members of the Ling lab and I am grateful for all their help and friendship. I thank my committee members, Dr. Ross MacGillivray and Dr. Natalie Strynadka, for their suggestions, encouragement and support. Without them, I am sure my Ph.D. would have taken much longer. To my parents, I thank them for always believing in me and letting me find my own way. Finally, I thank my wife, Ji-Yeon Hong, for giving so much love and support during the tribulations of graduate school. She is always there to lift me up and keep me going. I Introduction 1.1 — ABC transporters The largest known family of transmembrane proteins is the superfamily of ATP-binding cassette (ABC) transport systems which are found in prokaryotes, eukaryotes and archeabacteria (9,17). Some of the first ABC transporters discovered in eukaryotes were: P- gycoprotein, a multidrug transporter that is over-expressed in tumors resistant to chemotherapy (4); the products of the white and brown genes in Drosophila melanogaster, which transport pigments (13); and the cystic fibrosis transmembrane conductance regulator (CFTR) which functions as a chloride channel (31). Currently there are 49 members of the human family of ABC transporters annotated in the human genome, several of which have been implicated in human ailments (2,11,12,42). ABC transporters all contain a highly conserved ATP-binding cassette or nucleotide binding domain (NDB) which consists of a Walker A (G-X2-G-X-G-K-S/T-T/S-X4- hydrophobic) and a Walker B (R-X-hydrophobic2-X2-P/T/S/A-X-hydrophobic4-D-E-A/P/C- T-S/T/A-A/G-hydrophobic-D) motif, which are similar to those originally described by Walker (41).
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