Functional reconstitution of the human lysosomal peptide transporter TAPL reveals the substrate specificity [Elektronische Ressource] / von Chenguang Zhao

Chenguang

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Functional reconstitution
of the human lysosomal peptide transporter TAPL
reveals the substrate specificity
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main
von Chenguang Zhao
aus Huhehaote/China
Frankfurt am Main, 2009
(D30)Vom Fachbereich Biochemie, Chemie und Pharmazie der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.
Dekan: Prof. Dr. Dieter Steinhilber
1. Gutacher: PD. Dr. Rupert Abele
2. Gutachter: Prof. Dr. Clemens Glaubitz
Datum der Disputation:Teile der vorliegenden Arbeit wurden veröffentlicht in:
Zhao C, Haase W, Tampé R, Abele R.
Peptide Specificity and Lipid Activation of the Lysosomal Transport Complex ABCB9
(TAPL).
J Biol Chem (2008) 283: 17083-17091
Zhao C, Tampé R, Abele R.
TAP and TAP-like — brothers in arms?
Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 372: 444–450Table of Contents
Table of Contents
Deutsche Zusammenfassung ------------------------------------------------------------------------------------------- 4
Abstract--------------------------------------------------------------------------------------------------------------------- 8
1 Introduction ------------------------------------------------------------------------------------------------------------10
1.1 ABC Transporters -----------------------------------------------------------------------------------------------10
1.1.1 Structural organization -------------------------------------------------------------------------------------- 10
1.1.2 Transport mechanism ---------------------------------------------------------------------------------------- 17
1.2 Transporter associated with antigen processing-like (TAP-like or TAPL) --------------------------18
1.2.1 Phylogenetic relationship and gene organization------------------------------------------------------ 18
1.2.2 Topology model and homodimerization ----------------------------------------------------------------- 20
1.2.3 TAPL functions as a peptide transporter --------------------------------------------------------------- 22
1.2.4 Nucleotide-dependent peptide transport ---------------------------------------------------------------- 23
1.2.5 Tissue distribution and cellular localization------------------------------------------------------------ 24
1.2.6 Physiological function ---------------------------------------------------------------------------------------- 25
1.3 Objective -----------------------------------------------------------------------------------------------------------27
2 Materials ----------------------------------------------------------------------------------------------------------------28
2.1 Chemicals ----------------------------------------------------------------------------------------------------------28
2.2 Primers -------------------------------------------------------------------------------------------------------------31
2.3 Peptides ------------------------------------------------------------------------------------------------------------33
3 Methods------------------------------------------------------------------------------------------------------------------34
3.1 Molecular cloning ------------------------------------------------------------------------------------------------34
3.1.1 Plasmid DNA preparation----------------------------------------------------------------------------------- 34
3.1.2 Ligase chain reaction for site-directed in vitro mutagenesis ---------------------------------------- 34
3.1.3 Plasmid DNA restriction analysis ------------------------------------------------------------------------- 35
3.1.4 Subcloning the gene of interest by restriction digestion and ligation ---------------------------- 36
3.1.5 Subcloning the cDNAs of Haf-4 and Haf-9 by PCR -------------------------------------------------- 36
3.1.6 Isolation of the recombinant Bacmid DNA ------------------------------------------------------------- 37
3.2 Microbiological techniques-------------------------------------------------------------------------------------37
3.2.1 Bacterial culture ----------------------------------------------------------------------------------------------- 37
3.2.2 Preparation of competent cells ----------------------------------------------------------------------------- 38
3.2.3 Transformation of plasmid DNA into chemical competent bacterial cells---------------------- 38
3.3 Cell biology techniques------------------------------------------------------------------------------------------39
3.3.1 Thawing and freezing of Sf9 insect cells ----------------------------------------------------------------- 39
3.3.2 Sf9 insect cells culture ---------------------------------------------------------------------------------------- 40
3.3.3 Transfecting Sf9 cells with recombinant Bacmid DNA ---------------------------------------------- 40
3.3.4 Amplification of the recombinant baculovirus--------------------------------------------------------- 40
3.3.5 Virus titer determination ------------------------------------------------------------------------------------ 41
3.3.6 Recombinant membrane protein expression ----------------------------------------------------------- 41
3.4 General biochemical methods ---------------------------------------------------------------------------------42
1Table of Contents
3.4.1 Protein concentration determination--------------------------------------------------------------------- 42
3.4.2 SDS-PAGE ------------------------------------------------------------------------------------------------------ 43
3.4.3 Immunoblotting------------------------------------------------------------------------------------------------ 43
3.5 Biochemical assays for TAPL ---------------------------------------------------------------------------------45
3.5.1 Peptide labeling with 5-iodoacetamidofluorescein ---------------------------------------------------- 45
1253.5.2 Peptide labeling with Na I--------------------------------------------------------------------------------- 45
3.5.3 Membrane preparation -------------------------------------------------------------------------------------- 46
3.5.4 Membrane solubilization ------------------------------------------------------------------------------------ 46
3.5.5 Determination of critical micelle concentration ------------------------------------------------------- 47
3.5.6 Purification------------------------------------------------------------------------------------------------------ 47
3.5.7 Gel filtration ---------------------------------------------------------------------------------------------------- 48
3.5.8 Blue native PAGE --------------------------------------------------------------------------------------------- 48
3.5.9 Reconstitution -------------------------------------------------------------------------------------------------- 50
3.5.10 Freeze fracture electron microscopy -------------------------------------------------------------------- 51
3.5.11 ATPase activity assay --------------------------------------------------------------------------------------- 51
3.5.12 Peptide transport assay------------------------------------------------------------------------------------- 52
3.5.13 Peptide transport assay (glycosylation assay)--------------------------------------------------------- 52
3.5.14 Co-immunoprecipitation ----------------------------------------------------------------------------------- 53
4 Results -------------------------------------------------------------------------------------------------------------------54
4.1 Functional expression of TAPL -------------------------------------------------------------------------------54
4.2 Solubilization of TAPL------------------------------------------------------------------------------------------55
4.3 Purification of TAPL--------------------------------------------------------------------------------------------57
4.4 Oligomeric status-------------------------------------------------------------------------------------------------59
4.5 Functional reconstitution of TAPL---------------------------------------------------------------------------61
4.6 Lipid activation of TAPL---------------------------------------------------------------------------------------64
4.7 Key positions for substrate recognition ---------------------------------------------------------------------66
4.8 Sequence specificity of TAPL----------------------------------------------------------------------------------67
4.9 ATPase activity ---------------------------------------------------------------------------------------------------70
4.10 Functional solubilization and purification of TAPL by dodecylmaltoside -------------------------72
4.11 Functional expression of TAPL cysteine-less mutant and single cysteine mutants---------------77
4.12 Characterization of TAPL orthologs from Caenorhabditis elegans----------------------------------81
4.12.1 Expression of Haf-4 and Haf-9 in Sf9 insect cells---------------------------------------------------- 81
4.12.2 Haf-4 and Haf-9 are ATP-dependent peptide transporters --------------------------------------- 82
4.12.3 Interaction between Haf-4 and Haf-9------------------------------------------------------------------- 86
5 Discussions -------------------------------------------------------------