Brigham Young University BYU ScholarsArchive Theses and Dissertations 2014-04-01 The Roles of Phosducin-Like Protein 1 and Programmed Cell Death Protein 5 as Molecular Co-Chaperones of the Cytosolic Chaperonin Complex Christopher M. Tracy Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Chemistry Commons BYU ScholarsArchive Citation Tracy, Christopher M., "The Roles of Phosducin-Like Protein 1 and Programmed Cell Death Protein 5 as Molecular Co-Chaperones of the Cytosolic Chaperonin Complex" (2014). Theses and Dissertations. 5277. https://scholarsarchive.byu.edu/etd/5277 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. The Roles of Phosducin-Like Protein 1 and Programmed Cell Death Protein 5 as Molecular Co-Chaperones of the Cytosolic Chaperonin Complex Christopher M. Tracy A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Barry M. Willardson, Chair Allen R. Buskirk Gregory F. Burton Paul B. Savage John T. Prince Department of Chemistry and Biochemistry Brigham Young University April 2014 Copyright © 2014 Christopher M. Tracy All Rights Reserved ABSTRACT The Roles of Phosducin-Like Protein 1 and Programmed Cell Death Protein 5 as Molecular Co-Chaperones of the Cytosolic Chaperonin Complex Christopher M. Tracy Department of Chemistry and Biochemistry, BYU Doctor of Philosophy A fundamental question in biology is how proteins, which are synthesized by the ribosome as a linear sequence of amino acids, fold into their native functional state. Many proteins require the assistance of molecular chaperones to maneuver through the folding process to protect them from aggregation and to help them reach their native state in the very concentrated protein environment of the cell. This study focuses on the roles of Phosducin-like Protein 1 (PhLP1) and Programmed Cell Death Protein 5 (PDCD5) as molecular co-chaperones of the Cytosolic Chaperonin Complex (CCT). Signaling in retinal photoreceptors is mediated by canonical G protein pathways. Previous in vitro studies have demonstrated that Gβ subunits rely on CCT and its co-chaperone PhLP1 to fold and assemble into Gβγ and RGS-Gβ5 heterodimers. The importance of PhLP1 in the assembly process was first demonstrated in vivo in a retinal rod photoreceptor-specific deletion of PhLP1. To test whether this mechanism applied to other cell types, we prepared a second mouse line that specifically disrupts the PhLP1 gene in cone photoreceptor cells and measured the effects on G-protein expression and cone visual signal transduction. In PhLP1 depleted cones, Gt2 and RGS9-Gβ5 levels were dramatically reduced, resulting a 60-fold decrease in cone sensitivity and a 50-fold increase in cone photoresponse recovery time. These results demonstrate a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, underlining the significance of PhLP1/CCT-mediated folding in G protein signaling. PDCD5 has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for PhLP1 binding partners revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5-CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 binds the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding. Key words: Chaperonin, chaperone, G-protein signaling, phosducin-like protein, CCT, PhLP1, PDCD5, apoptosis ACKNOWLEDGEMENTS I wish to acknowledge all of the many people that contributed to this work. I especially want to thank Dr. Barry Willardson for the leadership and direction he provided me as I performed this research. I also thank my committee for their help and advice, as well as the Department of Chemistry and Biochemistry and the University for allowing me to pursue my doctorate here. I especially want to thank the wonderful people that I have worked with in the Willardson lab, particularly Amy Gray, Rebecca Plimpton, Jeffrey Lai, Tanner Shaw, and Devon Blake who directly contributed to this work. I also want to thank our collaborators Jorge Cuellar, Jose Valpuesta, Sasha Kolesnikov, and Vladimir Kefalov. Most importantly I want to thank my wife Miriam for her love, support, and dedication to our family and our three children Brigham, Andalynn, and Alora. TABLE OF CONTENTS List of Tables and Figures ........................................................................................................ vii Abbreviations .......................................................................................................................... viii Chapter 1: Chaperone-Mediated Assembly of G-Protein Complexes ...........................................1 Summary ...............................................................................................................................1 Introduction ...........................................................................................................................1 Chaperonins ....................................................................................................................2 G Protein Signaling .........................................................................................................3 Assembly of the Gβγ Dimer.............................................................................................6 Chaperone-mediated Assembly of G protein complexes ..................................................7 Role of PhLP1 phosphorylation in Gβγ assembly .......................................................... 10 Mechanism of Gβ association with Gγ ........................................................................... 11 Association of Gβγ with Gα ........................................................................................... 12 Specificity of PhLP1-mediated Gβγ assembly ................................................................ 14 Assembly of the RGS-Gβ5 dimer ................................................................................... 16 Conclusion .................................................................................................................... 18 Chapter 2: Characterization of Phosducin-like Protein 1 Gene Deletion in Cone Cells of the Mouse Retina ............................................................................................................................ 20 Summary ............................................................................................................................. 20 Introduction ......................................................................................................................... 20 Experimental Procedures ..................................................................................................... 24 Development of cone phlp1 gene deletion...................................................................... 24 Antibodies ..................................................................................................................... 25 Immunohistochemistry and assessment of photoreceptor degeneration .......................... 25 Determination of retinal protein expression.................................................................... 26 iv Quantitative RT-PCR .................................................................................................... 27 Assessment of the photo-response by Electroretinography ............................................. 28 Optomotor responses ..................................................................................................... 29 Transretinal ERG Recordings ........................................................................................ 30 Results ................................................................................................................................ 30 Confirmation of the cone-PhLP1 deletion ...................................................................... 30 Assessment of cone viability .......................................................................................... 32 PhLP1 deletion causes a decrease in cone Gt .................................................................. 32 PhLP1 deletion causes a decrease in cone RGS9-Gβ5 .................................................... 34 PhLP1 knockout results in decreased photopic phototransduction .................................. 36 PhLP1 knockout results in prolonged photo response recovery ...................................... 40 Discussion ..........................................................................................................................