The PX domain protein interaction network in yeast Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) der Fakultät für Chemie und Biowissenschaften der Universität Karlsruhe (TH) vorgelegte DISSERTATION von Dipl. Biol. Carolina S. Müller aus Buenos Aires Dekan: Prof. Dr. Manfred Kappes Referent: Dr. Nils Johnsson Korreferent: HD. Dr. Adam Bertl Tag der mündlichen Prüfung: 17.02.2005 I dedicate this work to my Parents and Alex TABLE OF CONTENTS Table of contents Introduction 1 Yeast as a model organism in proteome analysis 1 Protein-protein interactions 2 Protein Domains in Yeast 3 Classification of protein interaction domains 3 Phosphoinositides 5 Function 5 Structure 5 Biochemistry 6 Localization 7 Lipid Binding Domains 8 The PX domain 10 Function of PX domain containing proteins 10 PX domain structure and PI binding affinities 10 Yeast PX domain containing proteins 13 PX domain and protein-protein interactions 13 Lipid binding domains and protein-protein interactions 14 The PX-only proteins Grd19p and Ypt35p and their phenotypes 15 Aim of my PhD work 16 Project outline 16 Searching for interacting partners 16 Confirmation of obtained interactions via a 16 second independent method Mapping the interacting region 16 The Two-Hybrid System 17 Definition 17 Basic Principle of the classical Yeast-Two Hybrid System 17 Peptide Synthesis 18 SPOT synthesis technique 18 Analysis of protein- peptide contact sites based on SPOT synthesis 19 TABLE OF CONTENTS Experimental procedures 21 Yeast two-hybrid assay high-throughput screening 21 Interaction mating using robotic procedures 22 Bait and Prey construction 24 Vectors 25 1st Round PCR 28 2nd Round PCR 28 Scoring of yeast two-hybrid high-throughput PX interactions 29 Yeast genomic DNA preparation 30 Yeast Strains 31 Cloning by recombination and Yeast transformation 31 Yeast Media 33 GST-PX Domain fusion proteins 35 GST-Fusion Proteins Expression 36 Preparation and Transformation of “Ultra Competent” E.coli Cells 38 SDS PAGE 40 Coomassie Blue Staining of Acrylamide Gels 40 In vitro transcription and translation using the TNT T7 42 Quick for PCR DNA GST Pull-Down 44 Site directed mutagenesis using the QuickChange Method 45 Lipid overlay assay 49 Peptide Synthesis 50 β−Alanine membranes 50 Peptide Synthesis 51 TFA removal of all side chain protecting groups 52 (for β-Alanine membranes) TFA removal of all side chain protecting groups 52 (for PEG membranes = Intavis membranes) Positive control peptides 52 Protein Incubation 53 Pulse + Chase Immunoprecipitation 54 Yeast protein extracts 56 Western Blot Transfer (semi-dry) 57 Western Blot Detection 58 Lucifer yellow assay 59 TABLE OF CONTENTS Results 61 Yeast two-hybrid data 61 Full-length vs. domain interactions 66 The nature of PX protein interactors 68 In vitro data 69 PX domains interact with members of the Yip1 family in vitro 69 Comparison of two-hybrid and in vitro binding data 71 Ypt35-PX binds to Yip1 and Yif1 N-Terminus 71 PI-binding site is different from putative protein-protein interaction sites 72 Peptide arrays 74 Yip1 family members 75 Experimental setup 75 Recognizing interacting regions 75 Mapping to the single amino acid with alanine scans 75 PX domains bind to basic peptides at the N-terminus of Yip1 family 86 members and their intermembrane loops Binding site conservation among Yip1 family members 86 Differences between PX domains 86 Non-Yip1 family members 90 Rrn10p peptides incubated with Snx42-PX GST fusion protein 90 RBD2 peptides incubated with Grd19p GST fusion protein 91 Ylr031w peptides incubated with Grd19p GST fusion protein 92 Ynl086w peptides incubated with Grd19p GST fusion protein 92 Yip1p HIITKTKM motif analysis on Ypt35-PX interaction 93 Temperature-sensitive mutant Yip1-2 (G175E) is able to interact 94 in vitro but not in vivo with PX proteins and domains TABLE OF CONTENTS Discussion 97 PI binding domains as protein interaction domains 97 The nature of PX protein interactors 97 False negatives 97 Localization of PX interactors 97 Nuclear PX interactions 98 PX domain interaction map: a pool of new information 99 False negatives 99 New high confidence interactions 99 Vam7p/Vam7 PX – Hsv2p 99 Vam7p/Vam7 PX – Bsp1p 99 Rbd2p 100 Akr2p 100 Yip1 family members 101 Yip1 family member’s localization and function 101 Yip1 family member’s interactors localization 101 PX domain proteins in the Cvt pathway 103 Mapping the interacting region on Yip1 family members 105 The PI-binding site is different from the protein-protein 106 interaction site Structural evidence for possible simultaneous binding of 106 lipids and proteins Simultaneous lipid and protein binding and phospholipid 107 distribution in the cell Peptide arrays Analysing different membranes 108 Distribution of Yip1 family member binding peptides 108 PX domain interacting motif on Yip1 family members 108 Analysis of the HITKTKM Yip1 motif on Ypt35-PX interaction 111 Mutational analysis 111 Competition assay 112 PX domain´s interaction site 112 The biological function of PX-Yip interactions 113 Pulse-Chase labelling of Kex2p 114 Yip1p temperature sensitive mutant interacts with Ypt35-PX 116 in vitro but not in vivo TABLE OF CONTENTS Open questions, strategies, and future perspectives 118 How do PX domains and proteins contribute to Yip1 family 118 function? Yip1p/Yif1p 118 Yip4p/Yip5p 119 Can we find out more by about PX proteins looking at 119 their genetic interactions? Do PX domains bind simultaneously lipids and proteins? 119 Which surfaces interact in Yip-PX interactions? 120 Appendix 121 Abbreviations 129 References 131 Summary 139 Zusammenfassung 141 INTRODUCTION Introduction Yeast as a model organism in proteome analysis Budding yeast was the first eukaryotic organism to be completely sequenced (Goffeau et al. 1996). It has a relative small genome size (12 Mb compared to 3,2 Gb in humans) and contains 16 chromosomes encoding approximately 6000 genes. The fact that introns in yeast genes are rare (5% of genes) makes in silico gene prediction relatively straight forward and systematic gene nomenclature simplifies data comparison. Moreover, despite that this unicellular eukaryotic organism is only one tenth the size of a white blood cell, many of the cellular functions of higher species are present in yeast. S.cerevisiae has nearly 1800 genes homologous to human genes of which approximately 200 are related to human disease genes including 23 related to cancer (Rubin et al. 2000). For these reasons and the fact that yeast cells allow efficient homologous DNA recombination, S.cerevisiae has served as the prime model organism for post-genome functional analysis. Many systematic approaches have been used to study various aspects of the function and dynamics of the genome ranging from the analysis of deletion phenotypes to biochemical activities (Table 1). The yeast community has the most complete repertoire of physical resources: chips, reagents, plasmids, and mutant strains, covering the whole genome of this organism. This reflects the power of forward and reverse genetic approaches in yeast and hence yeast is certainly the best understood eukaryote. Project References Systematic gene deletion (Wach et al. 1994; EUROFAN I (European Functional Analysis Network) 1995-1999 Oliver 1996) 800 open reading frames were deleted and functionally analysed Systematic gene deletion The Saccharomyces genome deletion project (EUROFANII): (Winzeler et al. Creation of a complete set of bar-coded yeast deletion strains 1999; Giaever et (1996-2002) & phenotypic analysis of sporulation and mating al. 2002) efficiency Random transposon mutagenesis TRIPLES (Transposon-Insertion Phenotypes, Localization and (Ross-Macdonald Expression in Saccharomyces). >20 000 yeast mutants were et al. 1999) generated by random transposon mutagenesis and their phenotypes and localization were subsequently assigned to genes by sequencing. Large-scale yeast two-hybrid studies of protein-protein interactions (Uetz et al. 2000; (yeast protein interactome) Ito et al. 2001) Systematic identification of protein complexes in yeast by mass (Gavin et al. 2002; spectrometry Ho et al. 2002) - 1 - INTRODUCTION Project References (Hughes et al. Microarray gene expression studies at different conditions 2000) Global analysis of protein expression in yeast (Ghaemmaghami (quantitative immunobloting) et al. 2003) Yeast genetic interaction network (double deletions) (Tong et al. 2004) Yeast proteome subcellular localization (GFP and (Kumar et al. immunolocalization) 2002; Huh et al. 2003) 100 essential genes of unknown function have been fused to a (Sanchez-Diaz et 'heat-inducible-degron' cassette that targets the protein for al. 2004), proteolysis at 37 degrees (degron strains). (Kanemaki et al. Integrated analysis (affinity purification and mass spectrometry, two- 2003), (Hazbun et hybrid analysis, fluorescence microscopy and structure prediction al. 2003) methodology) of 100 unknown essential genes. Table 1: Systematic approaches to functional analysis in S.cerevisiae Mark Johnston, president of the Genetics Society of America, predicted at the Yeast Genetics and Molecular Biology meeting in July 2003 in Gothenburg, that the organism's 6,000 genes will be “known” (i.e. annotated) by 2007. This was based on the annotations in the Yeast Proteome Database which have followed a remarkably linear upward trajectory, reaching 4,679 functionally annotated genes in July 2003. According to the Saccharomyces Genome Database (Dwight et al. 2004) still 1677 (Sept 2004) genes have