Padi: A novel subunit of the 26S proteasome in fission yeast Mary Penney Thesis presented for the Degree of Doctor of Philosophy at the University of Edinburgh 1998 Quotations "Trapped like rodentiol" "It's time we face reality, my friends. We're not exactly rocket scientists." Acknowledgements The last three years have had there ups and downs, and without the help of quite a number of people there would no doubt have been more downs than ups. As it is, here is my thesis, finished, and there were far more ups than downs. I would have got absolutely nowhere without all the past and present pombeites, Cohn Gordon, Caroline Wilkinson, Gordon McGurk, Mairi Wallace, Paul Kersey, Susan Thomson, and Itaru Samejima, for their help and support. Particularly, thank you to my supervisor Cohn Gordon for letting me have so many holidays, especially the now infamous Africa trip; Caroline Wilkinson, a.k.a. Brown Eyed Girl a.k.a. Lab Chum, for so many patient re-readings of paper and thesis drafts, for being my travelling companion in Africa and especially for being a great friend and introducing me to the joys of cricket. Roll on the boxing day test at the MCG!!; Gordon McGurk, a.k.a. Blue eyed boy. What can I say Gordon? I can't beat your acknowledgement to me in your thesis, your name is hardly as amusing as mine is it? You are a great bloke too and without your studious example I would never have known how the perfect Ph.D. student should act! There are also many other people on C4 without who's support, encouragement and friendship I would never have got this far and without whom life would certainly have been very dull, thank you, Eleanor Simpson, Siobhan Jordan, Peter Budd, Marina McKenzie, Julia Bell, Jane Alfred, Ruth Suffolk, Ian Jackson, Katerina Manolakou and late arrival Peter Currie. In addition, how could I have functioned for 3 years without the help of photography? Thank you Norman and Douglas, but especially Sandy Bruce. Everyone loved the pink boomerang slide and the cheeky little Padi subunit! My family and friends outside science have also been a great source of encouragement. You probably never understood a word I said about experiments, but I'm glad you asked and listened. Lastly, Kieran Jacka, thank you for your love and support, for being here with me and for caring. Words on paper aren't enough. I'll thank properly in person in Hobart. Mary iv ABSTRACT Mutations in the fission yeast genes mts5-1 and mbc]-1 were isolated in a screen for Schizosaccharomyces pombe (S.pombe) mutants that are both resistant to the microtubule destabilising drug methybenzylcarbamylate (MBCR) and temperature sensitive (t.s.) for growth. This screen has so far been specific for mutations in genes encoding subunits of the 26S proteasome (Gordon et al., 1993). This study shows that these strains contain mutations in the pad1 and crm] genes respectively. Crm] and pad] have previously been shown to be positive and negative regulators respectively of the AP-1 transcription factor Papi (Toda et al., 1992, Shimanuki et al., 1995), the S. pombe homologue of the mammalian AP-1 transcription factors fos and fun, which is involved in the transcription of multidrug resistance genes (Shimanuki et al., 1995). The mts5-1 (pad]-]) strain has a metaphase arrest phenotype and an increased level of high molecular weight ubiquitinated proteins when incubated at the restrictive temperature. This is identical to the mts2-1 (Gordon et al., 1993) and mts3-1 (Gordon et al., 1996) mutants isolated in the same screen and which have been shown to encode subunits of the 26S proteasome. This study reclassifies pad]' as a subunit of the 26S proteasome, 'and data is provided which shows genetic interactions between Padi and three other subunits of the 26S proteasome, Mts3 (Gordon et al., 1996), Mts4 (Wilkinson et al., 1997) and Pus (C. Wilkinson pers. comm.). A putative function for the 19S cap subunit Padi as an isopeptidase is also investigated. Crml has been implicated in MDR through Papi, since Papi is responsible for the transcription of genes involved in resistance to a wide variety of drugs. 26S proteasome mutants are also shown to be resistant to the same range of drugs as the mbcl-] (cnn]-I) mutant, but to a lower level, and that papli\ cells are sensitive to MBC (MBCS). 26S proteasome mutants are shown to have elevated levels of Papi when incubated at the permissive temperature indicating that this protein is not being degraded as efficiently as in wild type cells. A c.s. cnn] mutant has been shown to over-express a non-essential 25KDa protein that has been shown to be a downstream UV target of Papi (Adachi and Yanagida, 1989). This protein is also shown to be over expressed in S.pombe proteasome mutants and cnn]-1. paplA 26S proteasome double mutants are t.s. and MBCS. This is consistent with the 26S proteasome being involved in the degradation of Papi and hence involved in pleiotropic multi-drug resistance. Vi Abbreviations All units used in this thesis are Standard International (SI) units A Adenine WX amino acds Abs absorbance Ac Acetate ade adenine APC anaphase promoting complex Arg Arginine ARS autonomously replicating sequence 3-AT 3-amino triazole ATP adenosine triphosphate BFA brefeldin A BSA bovine serum albumin C cytosine C.albicans Candida albicans C.maltosa Candida maltosa C-terminal carboxyl-terminal CDC cell division control CDK cyclin dependent kinase cDNA complementary deoxyribonucleic acid CVFR cystic fibrosis transmembrane conductance regulator C.S. cold sensitive DAPI 4,6-diamidino-2-phenylindole dATP deoxyadenosine triphosphate dCTP deoxycytosine triphosphate dGTP deoxyguanosine triphosphate dH20 distilled water DMF dimethyl formamide DMSO dimethyl sulphoxide DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DUB deubiquitinating enzyme El ubiquitin activating enxyme E2 ubiquitin conjugating enzyme E3 ubiquitin protein ligase E. co/i Escherichia co/i EDTA ethylenediamine tetra-acetic acid disodium salt Vii EGTA ethylene glycol-bis(3aminoethyl ether)N,N,N' ,N' tetra acetic acid EM electron microscopy EMM Edinburgh minimal medium ER endoplasmic reticulum EtOH ethanol FACS fluorescent activated cell sorting FITC fluorescein isothiocyanate G guanosine GFP green fluorescent protein Glu glutamic acid Gin glutamine Gly giycine GST glutathione S transferase HA Haemaglutinin His histidine IPTG isopropyithio-3-D-thiogalactopyranosidase kb kilobase pairs KDa kilodaltons Leu leucine LMB leptomycin B Lys lysine MALDI matrix assisted laser desorption (mass spectrometry) MBC methyl benzimidazole-2-yl carbamyiate MBCR MBC resistant MCP multicatalytic proteinase MDR multidrug resistant ME malt extract Met methionine MgCl2 magnesium chloride MHC major histocompatibility complex min minutes Mr molecular mass mRNA messenger RNA MTOC microtubule organising centre mts MBC-resistant temperature sensitive NaOAc sodium acetate NLS nuclear localisation signal nmt no message thiamine VIII NPD non parental ditype NPS nuclear pore complex N-terminal amino-terminal OD optical density oligo oligonucleotide ORF open reading frame PA proteasome activator PAGE polyacrilamide gel electrophoresis PBS phosphate buffered saline PCD programmed cell death PCR polymerase chain reaction PD parental ditype PEG polyethylene glycol pers. comm. Personal communication PFA paraformaldehyde Phe phenylalanine PT propidium iodide PKA protein kinase A PNK polynucleotide kinase RNA ribonucleic acid rpm revolutions per minute rt room temperature s second(s) S. cerevisiae Saccharomyces cerevisae SDS sodium dodecyl sulphate Ser serine snRNA small nuclear ribonucleic acid SPB spindle pole body S.pombe Schizosaccharomyces pombe T thymine TE Tris:EDTA Thr threonine Tm melting temperature tRNA transfer ribonucleic acid Trp tryptophan TPR tetratrico peptide repeat TT tetra type Tyr tyrosine ix t.s. temperature sensitive ub ubiquitin UBC ubiquitin-conjugating enzyme UBP ubiquitin specific protease UV ultraviolet ura uracil X-Gal 5-bromo-4-chloro-3-indolyl-13-D-galactosidase YE yeast extract YPD yeast complete media gv -t.i+,- c Title Page Quotations ii Declaration iii Acknowledgements iv Abstract v Abbreviations vii Table of Contents xi List of Figures xix Table of Contents Chapter 1 Introduction 1 1.1 ATP-dependent proteolysis and its functions 2 1.1.1 Cellular proteolysis by the 26S proteasome 2 1. 1.2 The ubiquitin pathway for protein degradation 3 1.1.2.1 Selection of proteins for degradation 3 1.1.2.2 Ubiquitin 5 1.1.2.3 Intracellular protein degradation and Ub chains 5 1.1.3 Enzymes involved in ubiquitination 6 1.1.3.1 Ubiquitinating enzymes 6 1.1.3.2 De-ubiquitinating enzymes 7 1.2 The 26S proteasome 12 1.2.1 The 20S proteasome 12 1.2.1.1 Structure, biogenesis, enzymatic properties 12 1.2.1.2 Localisation of the 20S proteasome 15 1.2.1.3 In vivo functions 15 1.2.2 PA700, the 19S regulatory complex 17 1.2.2.1 ATPase subunts 17 1.2.2.2 Non-ATPase subunits 17 1.2.2.3 Functions of the 19S regulatory complex 20 1.2.3 Cellular functions of proteolysis by the 26S proteasome 21 1.2.3.1 Ubiquitinated substrate degradation 21 1.2.3.2 Programmed cell death and apoptosis 21 Xi 1.3 Control of the eukaryotic cell cycle 22 1.3.1 The fission yeast cell cycle 23 1.3.2 The metaphase to anaphase transition 25 1.3.2.1 Control of the metaphase to anaphase transition 27 1.3.2.2 Initiation of Anaphase 28 1.3.2.3 The anaphase promoting complex. 30 1.4 S.pombe MBCR t.s. mutant screen 32 1.4.1MBC 33 1.4.2 General drug resistance 33 1.4.3 Multidrug resistance 33 1.4.3.1 ABC transporters 34 1.4.3.2 MFS-type transporters 35 1.4.3.3 Transcription regulators 36 1.4.4 Drug resistance in S.pombe 38 1.4.4.1 The pap]' gene 38 1.4.4.2 The pad]' gene 38 1.4.4.3 The crmt gene 38 1.4.4.4 Drug transporters in S.pombe 40 1.5 Project aims 41 1.5.1 Characterisation of mts5-1 41 1.5.2 Characterisation of mbcl-1 42 Chapter 2 Materials and Methods 43 2.1 Commonly used reagents and buffers 44 2.2 Nucleic acid manipulations 46 2.2.1 Dissolving and storage 46 2.2.2 Extraction with phenol-chloroform 46 2.2.3 Precipitation of nucleic acids 46 2.2.4 Quantification of nucleic acids 46 2.2.5 Plasmid vectors 47 2.3 Molecular analysis of nucleic acids 51 2.3.