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REGULATORY PARTICLES of PROTEASOMES and DETERMINANTS of PROTEIN LEVEL REGULATION in the HALOARCHAEON Haloferax Volcanii

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REGULATORY PARTICLES of PROTEASOMES and DETERMINANTS of PROTEIN LEVEL REGULATION in the HALOARCHAEON Haloferax Volcanii REGULATORY PARTICLES OF PROTEASOMES AND DETERMINANTS OF PROTEIN LEVEL REGULATION IN THE HALOARCHAEON Haloferax volcanii By CHRISTOPHER J. REUTER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006 This document is dedicated in loving memory to my grandmother, Gladys Reuter, whom throughout my early schooling would reward me with a dollar for every “A” grade I received. This paper would have certainly been worth at least five. ACKNOWLEDGMENTS I would like to thank Dr. Julie Maupin-Furlow for allowing me to be in the position that I am today. By taking her undergraduate prokaryotic physiology class, I immediately gained a fascination for the world of molecular biology, and without her constant support and encouragement none of this would have been possible for me. I would also like to thank the members of my committee, Drs. L.O. Ingram, Madeline Rasche, Nemat Keyhani, and Anita Wright, for their time and patience. Although I rarely sought their counsel, I always knew their doors were always open. I am especially grateful to Dr. L.O. Ingram, whose guest presentation on microbial ethanol production in my undergraduate general microbiology class really opened up my mind to the power of microbes. I would also like to thank Donna Williams and the late Dr. Henry Aldrich for their assistance in microscopy and photography. Both, although I hardly knew them at the time, were so generous in their efforts involving my research. I also thank Drs. James Preston and K.T. Shanmugam and members of their labs for allowing me the use of their equipment and time. I would also like to thank Drs. William Gurley, Paul Shirk, Mark Donnelly, Arthur Horwich, Moshe Mevarech, and Jerry Eichler for gifts of plasmids, cell cultures, or use of instrumentation. I would like to thank former members of Dr. Maupin’s lab including Mark Ou and Drs. Heather Wilson, Steve Kaczowka, and LeeAnn Talarico-Blalock for taking the time to teach many laboratory techniques. I would especially like to thank Dr. Steve Kaczowka for many nights of scientific (and some not so scientific) conversations and for collaboration of some of the Pan work. I iii would also like to thank some of my fellow graduate students, with whom I became good friends, for their helpful discussions along the way, including Franz St. John, P. Aaron Kirkland and Drs. T. Brice Causey, Celeste Johnson-Causey, and Stuart Underwood. Other graduate students I would like to thank include Gosia Gil for creating Pan mutants and helpful discussion and Matt Humbard for computer assistance and helpful discussions. I would like to thank my very good friends Drs. Cassidy Sedacca and Emily Bille for their constant encouragement and partnership outside the lab. I would also like to thank my family members including my sister, Sarah Reuter, my grandfather Robert Reuter, and my Uncle Joseph Reuter for their encouragement in my pursuance of my Ph.D. I would especially like to thank my parents, Gregory and Debra Reuter, for without their support and love throughout my life, none of this would be possible. Last and most certainly not least, I would love to thank my wife and best friend, Jennifer Reuter, for being my biggest fan, and sometimes, hardest critic. She has instilled in me the confidence that has made me what I am today and has made many sacrifices that have allowed me to pursue this degree. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS............................................................................................ xi ABSTRACT.......................................................................................................................xv CHAPTER 1 LITERATURE REVIEW .............................................................................................1 Introduction...................................................................................................................1 ClpP Protease................................................................................................................1 HslUV Protease ............................................................................................................6 Lon Protease .................................................................................................................9 FtsH Protease..............................................................................................................12 20S Proteasome ..........................................................................................................14 AAA+ ATPases ..........................................................................................................20 AAA+ Domain ....................................................................................................21 Walker Motifs......................................................................................................22 Sensor Regions ....................................................................................................22 Second Region of Homology ..............................................................................23 Coiled-coil Domains............................................................................................23 Pore Motifs ..........................................................................................................24 Mechanistic Action of ATPases ..........................................................................25 Substrate Recognition.................................................................................................26 Ubiquitination......................................................................................................27 N-end Rule...........................................................................................................29 SsrA Tagging.......................................................................................................30 Other Modes of Proteolytic Substrate Recognition.............................................31 Green Fluorescent Protein (GFP) as A Model Substrate.....................................32 Research Objective .....................................................................................................36 2 ANALYSIS OF PROTEASOME-DEPENDENT PROTEOLYSIS IN Haloferax volcanii, USING SHORT-LIVED GREEN FLUORESCENT PROTEINS ..............39 v Introduction.................................................................................................................39 Materials and Methods ...............................................................................................41 Materials..............................................................................................................41 Strains, Media and Plasmids................................................................................41 DNA Purification and Transformation................................................................42 Protein Techniques and Immunoblot...................................................................43 Fluorescence Measurements of GFP Variants in Whole Cells ...........................44 Purification of EGFP and 20S Proteasomes from H. volcanii ............................45 Peptide Hydrolyzing Activity..............................................................................46 RNA Isolation and Analysis................................................................................46 Results and Discussion ...............................................................................................47 Functional Synthesis of an Archaeal GFP Reporter Protein ...............................47 C-terminal Modifications Reduce the Level of smRS-GFP Protein in vivo .......48 C-terminal Modifications Do Not Impact the Level of smRS-GFP-specific mRNA in H. volcanii .......................................................................................51 Clasto-lactacystin β-lactone as an in vivo Inhibitor of H. volcanii 20S Proteasomes .....................................................................................................51 Inhibition of 20S Proteasomes Enhances the Level of smRS-GFP-SsrA Protein in H. volcanii .......................................................................................53 Conclusion ..................................................................................................................54 3 DIFFERENTIAL POST-TRANSLATIONAL REGULATION OF GREEN FLUORESCENT PROTEIN IN THE HALOARCHAEON HALOFERAX VOLCANII IS DETERMINED IN PART BY ITS EXTREME CARBOXYL TERMINAL RESIDUE..............................................................................................67 Introduction.................................................................................................................67 Materials and Methods ...............................................................................................70 Materials..............................................................................................................70 Strains, Media, and Plasmids...............................................................................70
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