Durham E-Theses Toward the characterisation of the protein-protein interactions of the type II fatty acid synthase of Brassica napus. Honeyman, Gordon How to cite: Honeyman, Gordon (2004) Toward the characterisation of the protein-protein interactions of the type II fatty acid synthase of Brassica napus., Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/3072/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 Gordon Honeyman. Toward the characterisation of the protein~protein interactions of the type H fatty acid synthase of Bras sica nap us. Abstract. The availability of eDNA and antibodies for the proteins of type II fatty acid synthase (FAS) of Brassica napus allowed an investigation into the protein-protein interactions of the putative type II FAS complex. Using the yeast two-hybrid system, four components were assayed for interactions: acyl carrier protein (ACP), enoyl­ ACP reductase (ENR), oleoyl-ACP thioesterase (TE), and stearoyl-ACP desaturase (DES). Direct interactions were detected between DES and ENR. A possible, very weak, interaction may have been detected between TE and ENR. The relative affinity of DES for ENR was greater than that of TE for ENR. Both were much weaker than ENR homo-tetrameric interactions. No interaction was found between ACP and any other FAS enzyme tested by this technique. I Toward the characterisation of the protein-protein interactions of the type II fatty acid synthase of Bras sica nap us. Gordon Honeyman. A thesis presented for the degree of Doctor of Philosophy, University of Durham. Department of Biological Sciences. ;& @SEP 200~ August, 2004. II Table of Content§" AbstraCtoooooooooooooooooooooooooooooooooooooooooooooeooooooooooooooooooooooooooooooooooooooooooooooooooooooo:n: Titleaoeooooooooo~~>ooooGoooooooooaooooooooooDooooGJooooooooooooooooooooooooooooooooooooooooooaaoooaoaaoooooooooeoll TabI e of Con ten tse. •o•• ••••••••••••••••••• ····o········•o•• ••••••••••••••••••••••••••• o• ••••••••••• III In.de:x of FigureSooooooooooooooooooooooooooooooooooooooooooGoooooooooooooooooooooooooooooooooooooooo VN:n: Index: to TabI es •................................ o •••• o •••• o ••••••••••••••••••••••••••••••••••••••••• XII Abbreviation List., ............. •o •••••••••••••• o ••••••••••••• o. •••oo. o o ••••••• XVII DecJaratiODoooooeoooooooooooooeoooooooooooooooooooooooooooooooooooooooooooooooooooe~ooooeoooooooo o ooooXX AckDOWledgemeDtSoooeooooooooooooooeoooooooooooooooeoooeoooooooeooooooooooooooooooeo o o o ooo o .XXI Chapter 1. Introduction....................................................................................... ! 1.1 De novo fatty acid synthesis of higher plants................................................... ! 1.1.1. Initiation of chain growth ............................................................................... 2 1.1.2. Acetyl-CoA carboxylase - The committed step ............................................. 2 1.1.3. Acyl carrier protein ........................................................................................ 4 1.1.4. The condensation reactions ............................................................................ 5 1.1.5. Fatty acid desaturation and termination of synthesis ...................................... 6 1.1.6. Lipid assembly pathways ............................................................................. 11 1.2. Macromolecular organisation and compartmentalisation of metabolism. ............................................................................................................................. 13 1.2.1. Macromolecular crowding (the excluded volume effect). .. ........................ 15 III 1.2.2. Multienzyme complexes ............................................................................. .20 1.2.3. Metabolic channeling .................................................................................. 23 1.3. lEvi«lJell]ce tllunt nue comp<mell]lts of type H lF A§ are associate«ll witllnill] a mllllHtnell]zyme compHeili: ........................................................................27 1.3 .1. Metabolic channeling of type II F AS ......................................................... 27 1.3.2. The co-purification of components of type II FAS of B. napus ............... 33 1.3.3 The interactions of E. coli ACP with other fatty acid synthase enzymes ..... 37 V!l. Tllne in vitro all]aHysis of proteill]-]pJrOteill] nll]teradiom ••••••••••••••••••••••••••••• AS 1.5. Aims of the tllnesis ............................................................................................ 49 CllnatJP>teD." .2. MateD."natll anull Metllnl[])dl§ ...............................................................so 2.1l Yea.st two-hybrid. system ............................................................... 50 2.1.1. Antibiotics, X-gal stocks and media ............................................................ 50 2.12. Plasmids ............. , .......................................................................................... 52 2.1.3. Bacterial strains ............................................................................................. 54 2.1.4. Yeast strains .................................................................................................. 54 2.1.5. Cloning of the eDNA of ACP, ENR, TE, and DES into vectors pAS2-l and pACT2 ................................................................................. 55 2.1.5a. DNA isolation ............................................................................................ 51 2.1.5b.Polymerase chain reaction (PCR) ............................................................... 57 2.1.5c. Restriction digest, ligations, and transformation of E. coli ........................ 59 IV 2.1.5d. DNA sequencing ........................................................................................ 60 2.1.6. Yeast transformations .................................................................................. 61 2.1.7 Interaction assay ............................................................................................ 63 2.1.8. Colony filter assay ....................................................................................... 63 2.1.9. Isolation of plasmid DNA from yeast (back extraction) ............................. 64 2.1.1 0. Protein extraction from yeast.. .................................................................... 65 2.1.11. Determination of protein concentration ...................................................... 66 2.1.12. Trichloroacetic acid (TCA) precipitation of yeast proteins .................... 66 2.1.13. SDS-Polyacrylamide gel electrophoresis (PAGE) of proteins ................... 66 2.1.14. Immunoblotting of SDS-PAGE seperated proteins ................................... 67 2.2. Bandshift analysis using isolated Brassica napus chloroplasts.................... 68 2.2.1. Plant Growth ................................................................................................. 68 2.2.2. Isolation of chloroplasts ............................................................................... 68 2.2.3. Oxygen electrode measurements of isolated Brassica napus chloroplasts . ............................................................................................................... ..... 70 2.2.4. Determination of chlorophyll content. ......................................................... 72 2.2.5. Immunoblotting to detect bandshifted proteins ................................. 73 2.3. Computer-based methods ................................................................................. 74 Chapter 3o ReSUI tsooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo 75 3.1. Analysis of protein-protein interactions of type H F AS of Brassica napus usillllg tllue yeast ... two ... Dtylt»~rirll system .......................................................................... 7.5 v 3.1.1. Cloning ............. o o ••••••••••••••••• o ••••••••• o •• 0 o 000 oo• •• o o o o o o o. o o 00 •oo o •o o o. o. 0 •• 80 3.1.la. Cloning: the prediction of the chloroplast-targetting peptide cleavage site of oleoyl-ACP thioesterase of Brassica napus . ..................................................... 81 3.1.1b. Primer design for cloning the eDNA of ACP, ENR, TE, and DES into the shuttle vectors ................................................................................. 85 3.1.1c. Preparation of DBD/ACP, DBD/ENR, DBD/TE, DBD/DES and AD/ACP, AD/ENR plasmid constructs ...................................................... 89 3.l.ld. Cloning: sequencing of constructs - identification of an anomaly in the Genbank database for the DNA sequence deposited for ACP of Brassica napus . .........................................................................