investigating the mechanism of escherichia coli min protein dynamics

INVESTIGATING THE MECHANISM OF ESCHERICHIA COLI MIN PROTEIN DYNAMICS by Laura L. Lackner Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Piet de Boer, PhD. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January 2006 UMI Number: 3193524 3193524 2006 UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 by ProQuest Information and Learning Company. CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Laura L. Lackner Lloyd A. Culp Jonatha M. Gott Patrick Viollier Pieter de Haseth Piet de Boer 8/4/2005 1 Table of Contents List of Tables 7 List of Figures 8 Acknowledgements 10 Abstract 11 Chapter 1: Introduction 13 Division proteins and the septal Ring 14 FtsZ and the Z-ring 15 ZipA and FtsA 17 FtsK 19 Additional essential septal ring components 19 Non-essential septal ring components 20 Spatial regulation of Z-ring assembly 21 Nucleoid Occlusion 22 The Min System 23 MinC, the division inhibitor 24 MinD 25 MinE 26 Min system dynamics spatially regulate the activity of MinC 27 Conservation of the Min proteins 29 Summary of this work 29 Chapter 2: ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro. 36 2 Summary 37 Introduction 38 Materials and Methods 44 Strains and plasmids 44 Functionality of tagged proteins in vivo 46 Purification of proteins 47 Preparation of phospholipid vesicles 49 Vesicle sedimentation assays 50 ATP hydrolysis 51 Results 52 Functionality of proteins used in this study 52 MinD associates with phospholipid membranes in an ATP- dependent fashion 54 Cooperative binding of MinD to phospholipid vesicles 57 MinE stimulates dissociation of MinD from phospholipid membrane 60 Role of nucleotide hydrolysis in MinD/MinE-dissociation from the membrane 62 MinD-dependent recruitment of the division inhibitor MinC to membrane 64 MinE-mediated release of both MinC and MinD from phospholipid vesicles 66 MinE-mediated specific release of MinC from MinC-MinD.ATPγS-membrane complexes 68 Discussion 69 Acknowledgements 77 3 Chapter 3: Roles of MinE domains in the regulation of Min protein-membrane dissociation in vitro and in vivo. 97 Summary 98 Introduction 99 Materials and Methods 105 Strains and plasmids 105 Protein purification 110 Gel filtration 111 Phospholipid preparation 112 ATPase assays 112 Sedimentation assays 113 Growth conditions and microscopy 114 Western analyses 115 Results 115 Purification of MinE deletion mutants 115 D MinE is necessary and sufficient to stimulate the ATPase activity of MinD 116 D MinE is necessary and sufficient to stimulate dissociation of MinD and MinC from the phospholipid membrane 116 Affinity of D MinE for the phospholipid membrane 119 D MinE removes Gfp-MinD from the membrane in vivo, but TS MinE is required to efficiently establish MinD dynamics 121 D MinE can induce formation of a D/E-tube, but fails to form an E-ring 124 Both D MinE and TS MinE are required to join an E-ring in wildtype cells 126 4 Discussion 129 Acknowledgements 135 Chapter 4: In vitro and in vivo characterization of the MinD Walker A motif mutant MinD K16R 147 Introduction 148 Materials and methods 150 Strains and plasmids 150 Protein purification 152 Lipid preparation, sedimentation and ATPase assays 153 Gel filtration 153 Growth conditions and microscopy 154 Yeast strains, plasmids, and two-hybrid analyses 155 Results 155 The MinD K16R -membrane interaction is nucleotide independent 155 MinE stimulates the dissociation of MinC from MinD K16R .ATP-membrane complexes but does not interfere with the MinD K16R -membrane interaction 157 MinE does not stimulate the ATPase activity of MinD K16R 159 MinD K16R forms spiral-like structures in the presence of MinE 160 The dimerization domain of MinE is required to form the MinD K16R structures 161 MinE exhibits a spiral-like accumulation in the presence of MinD K16R 162 The MinD K16R structures do not co-localize with MreB 163 5 Discussion 164 Acknowledgements 170 Chapter 5: Summary and Future Directions 182 The role of ATP in regulating the interactions of MinD with its many binding partners 183 How does MinE stimulate the ATPase activity of MinD? 186 The roles of monomeric and dimeric MinE 189 What role does MinE play in the formation of Min spirals? 192 Is the association of MinE with the membrane self-enhancing? 194 Are additional proteins required for MinDE dynamics? 195 A conserved mechanism for the oscillation of proteins in bacteria 197 Appendix A: Supplementary Data – FRET studies suggest that the MinD-MinD and MinD-MinE interactions are membrane dependent 201 Introduction 202 Materials and Methods 203 Strains and plasmids 203 Protein purification 204 Labeling proteins with AlexaFluor dyes 205 Phospholipid vesicle preparation 206 FRET assays 206 Results 207 Fluorescent labeling of MinD and MinE 207 FRET between A488-MinD and A594-MinD is lipid-dependent 208 FRET between A594-MinD and A488-MinE is 6 lipid-dependent 209 Discussion 210 Acknowledgements 211 Appendix B: Preliminary data – Attempts to establish Min protein dynamics in vitro 214 Introduction 215 Materials and methods 216 Protein purification 216 Trapping caged-ATP and Min proteins in lipid vesicles 216 Results 217 Discussion 218 Bibliography 222 7 List of Tables 2-1 In vivo functionality of His-tagged Min proteins 78 2-2 ATP- and Mg ++ -dependent association of H-MinD with phospholipid vesicles 79 2-3 Effect of prior co-purification with vesicles on the fraction of membrane-associating H-MinD 80 2-4 MinE-H stimulated dissociation of H-MinD from pre-decorated vesicles 81 3-1 Location of Gfp-MinD when co-expressed with the MinE fusions 136 3-2 Cellular distribution and oscillation cycle time of MinE-Gfp deletion mutants and Gfp-MinD in the presence of native MinCDE 137 4-1 In vivo and in vitro properties of MinD and MinD K16R 171 4-2 Yeast two-hybrid interactions 172 [...]... alters the cellular distribution of Gfp-MinDK16R 177 4-6 Localization of Gfp-MinDK16R in the presence of MinE deletion mutants 178 Localization of MinE-Gfp and MinE1-33-Gfp in the presence of MinDK16R 179 4-8 Localization of MreB and Gfp-MinDK16R 180 4-9 Localization of Gfp-MinDK16R in the absence of MreB 181 5-1 Structure of P furiosus MinD 200 A-1 MinD-MinD interactions monitored by FRET 212 A-2 MinD-MinE... regulating the 30 association and dissociation, respectively, of the Min proteins with the membrane To further characterize the role of MinE in Min protein dynamics, we investigated the contributions of each domain of MinE in the regulation of Min protein- membrane dissociation in vitro and in vivo (Chapter 3) We found that the monomeric DMinE domain is both required and sufficient to stimulate the ATPase... support In these models, the assumption that MinE forces the release MinD from the membrane is critical for the establishment of oscillation (Howard et al., 2001; Kruse, 2002; Meinhardt and de Boer, 2001) With the aim of gaining a better understanding of the roles ATP and MinE play in the reversible association of the Min proteins with the membrane, we explored the interactions between the Min proteins... Localization of MinE deletion mutants in the presence of MinD and co-localization of MinE deletion mutants and MinD 146 The association H-MinDK16R with phospholipid vesicles is nucleotide independent 173 4-2 Gel filtration analysis of MinD and MinDK16R 174 4-3 MinE stimulates the release of MinC but not MinDK16R from lipid vesicles 175 4-4 MinE does not stimulate the ATPase activity of MinDK16R 176 4-5 MinE... MinE stimulates the specific release of Gfp-MinC-H from H-MinD.ATPγS-decorated lipid vesicles 94 2-12 Model of ATP-driven MinCDE dynamics 95 3-1 MinE derivatives and their in vitro properties 138 3-2 Gel filtration analysis of purified MinE-M proteins 139 3-3 MinE1-33-M stimulates the ATPase activity of H-MinD in the presence of phospholipid vesicles 140 3-4 Min1 -33-M stimulates dissociation of H-MinD... recruits both MinC and MinE to the vesicles In addition, we found that MinE stimulates the dissociation of MinC, MinD, and itself from the vesicles Thus, ATP and MinE play critical roles in regulating the association and dissociation, respectively, of the Min proteins and the membrane 11 MinE has two known functional domains: the N-terminal anti-MinCD domain (DMinE) and the C-terminal topological specificity... Gfp-MinC-H from phospholipid vesicles 141 8 Nucleotide hydrolysis requirements for MinE1-33-M stimulated release of H-MinD and GFP-MinC-H from phospholipid vesicles 142 3-6 The N-terminus of MinE has affinity for phospholipid vesicles 143 3-7 Localization of GFP-MinD in the presence of the MinE deletion mutants 144 Cellular location of Gfp-MinD in the presence of increasing levels of MinE-M and MinE1-33-M... MinE-stimulated dissociation of MinD from vesicles 86 2-6 Stabilization of a MinE-MinD-membrane complex in the presence of ATPγS 87 2-7 Recruitment of MinE to MinD-decorated vesicles 88 2-8 Recruitment of MinC to MinD-decorated vesicles 89 2-9 Microscopy of phospholipid vesicles showing MinD-mediated recruitment and MinE-stimulated release of MinC 91 2-10 MinE-stimulated release of both MinC and MinD from phospholipid... constriction, the proteins of the septal ring function in concert to mediate the coordinated invagination of the three cell envelope layers: the inner membrane, the cell wall (the peptidoglycan layer), and the outer membrane Unfortunately, very little is known about the roles of many of the division proteins in this process FtsZ and the Z-ring FtsZ is the most widely conserved of the division proteins It... reveals that the protein dimerizes in an anti-parallel fashion The Nterminal domain is not shown in the structure but is predicted to be a nascent helix (King et al., 1999) Min system dynamics spatially regulate the activity of MinC The site of Z-ring assembly and, therefore, division is determined by the cellular distribution of MinC, which is regulated by both MinD and MinE In addition, MinD and MinE each . location of Gfp-MinD in the presence of increasing levels of MinE-M and MinE 1-33 -M 145 3-9 Localization of MinE deletion mutants in the presence of MinD and co-localization of MinE deletion. release of H-MinD and GFP-MinC-H from phospholipid vesicles 142 3-6 The N-terminus of MinE has affinity for phospholipid vesicles 143 3-7 Localization of GFP-MinD in the presence of the MinE. INVESTIGATING THE MECHANISM OF ESCHERICHIA COLI MIN PROTEIN DYNAMICS by Laura L. Lackner Submitted in partial fulfillment of the requirements for the degree of