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The Molecular Basis for Rapid Ca2+ Transport Across the Inner Mitochondrial Membrane

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The Molecular Basis for Rapid Ca2+ Transport Across the Inner Mitochondrial Membrane University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 The Molecular Basis For Rapid Ca2+ Transport Across The Inner Mitochondrial Membrane Riley J. Payne University of Pennsylvania Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Cell Biology Commons, and the Physiology Commons Recommended Citation Payne, Riley J., "The Molecular Basis For Rapid Ca2+ Transport Across The Inner Mitochondrial Membrane" (2019). Publicly Accessible Penn Dissertations. 3595. https://repository.upenn.edu/edissertations/3595 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3595 For more information, please contact [email protected]. The Molecular Basis For Rapid Ca2+ Transport Across The Inner Mitochondrial Membrane Abstract Ca2+ uptake by energized mitochondria regulates bioenergetics, apoptosis, and global Ca2+ signaling. Ca2+ entry into mitochondria is mediated by the Ca2+ uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The precise roles of these regulatory proteins and their relative stoichiometry in the complex have yet to be elucidated. MICU1 was proposed to be necessary for MCU activity whereas subsequent studies suggested a role for MICU1 and its paralog MICU2 in channel inhibition in the low-cytoplasmic Ca2+ ([Ca2+]c) regime, a mechanism referred to as “gatekeeping”. EMRE is required for MCU channel function and coupling of MICU1/2-mediated gatekeeping, but its stoichiometry relative to MCU in the native complex is unclear. We measured MCU activity over a wide range of quantitatively controlled and recorded [Ca2+]c to identify the regulatory function of the MICU1 and 2 and introduced mutations into Ca2+-binding sites in each protein to determine the mechanism by which [Ca2+]c controls channel activity. We then addressed the functional consequences of manipulating the relative stoichiometry of EMRE and MCU by introducing tagged MCU and EMRE in MCU/EMRE double-knockout cells to directly compare their expression and created a series of MCU-EMRE concatemers with multiple concatenated MCU subunits fused to EMRE, allowing us to “fix” their stoichiometry. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ uptake to mitochondria localized to nanodomains of high (>2 μM) [Ca2+]c. Furthermore, while incorporation of a single EMRE reconstitutes channel activity, additional EMRE subunits increase the [Ca2+]c-threshold required for relief of gatekeeping. Endogenous channels most likely contain two EMRE subunits which tether two MICU1/2-dimers to the complex. These findings have important implications for developing new therapies to target diseases arising from dysregulation of mitochondrial Ca2+-transport. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor James K. Foskett Keywords calcium transport, gatekeeping, ion channel, mitochondria, stoichiometry, uniporter Subject Categories Biochemistry | Cell Biology | Physiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3595 THE MOLECULAR BASIS FOR RAPID CA2+ TRANSPORT ACROSS THE INNER MITOCHONDRIAL MEMBRANE Riley J. Payne A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2019 Supervisor of Dissertation _________________________ J. Kevin Foskett , Ph.D. Isaac Ott Professor and Chair of Physiology Graduate Group Chairperson _________________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Roberto Dominguez, Ph.D. - William Maul Measey Presidential Professor of Physiology Joseph Baur, Ph.D. - Associate Professor of Physiology Marni Falk, MD - Executive Director, Mitochondrial Medicine Frontier Program Associate Professor of Pediatrics Benjamin L. Prosser, Ph.D. - Assistant Professor of Physiology THE MOLECULAR BASIS FOR RAPID CA2+ TRANSPORT ACROSS THE INNER MITOCHONDRIAL MEMBRANE COPYRIGHT 2019 Riley Joseph Payne DEDICATION For Brenda, who has been my support system, advocate and advisor throughout this journey and my grandmother, Marion, who taught me the value of persistence and patience. iii ACKNOWLEDGMENT I thank Dr. Vamsi Mootha for providing the MCU KO, MICU1 KO, MICU2 KO and EMRE KO cell lines. This work was supported by T32 HL 7954-16 (RP) and NIH R37 GM056238 (JKF). Special thanks to Dr. Horia Vais and Emily Fernandez Garcia for providing the unpublished data discussed in Chapter 4. iv ABSTRACT THE MOLECULAR BASIS FOR RAPID CA2+ TRANSPORT ACROSS THE INNER MITOCHONDRIAL MEMBRANE Riley J. Payne Prof. J. Kevin Foskett, Ph.D. Ca2+ uptake by energized mitochondria regulates bioenergetics, apoptosis, and global Ca2+ signaling. Ca2+ entry into mitochondria is mediated by the Ca2+ uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The precise roles of these regulatory proteins and their relative stoichiometry in the complex have yet to be elucidated. MICU1 was proposed to be necessary for MCU activity whereas subsequent studies suggested a role for MICU1 and its paralog MICU2 in channel inhibition in the low-cytoplasmic 2+ 2+ Ca ([Ca ]c) regime, a mechanism referred to as “gatekeeping”. EMRE is required for MCU channel function and coupling of MICU1/2-mediated gatekeeping, but its stoichiometry relative to MCU in the native complex is unclear. We measured MCU activity over a wide range of 2+ quantitatively controlled and recorded [Ca ]c to identify the regulatory function of the MICU1 and 2 and introduced mutations into Ca2+-binding sites in each protein to determine the mechanism 2+ by which [Ca ]c controls channel activity. We then addressed the functional consequences of manipulating the relative stoichiometry of EMRE and MCU by introducing tagged MCU and EMRE in MCU/EMRE double-knockout cells to directly compare their expression and created a series of MCU-EMRE concatemers with multiple concatenated MCU subunits fused to EMRE, allowing us to “fix” their stoichiometry. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ uptake to mitochondria localized to 2+ nanodomains of high (>2 µM) [Ca ]c. Furthermore, while incorporation of a single EMRE 2+ reconstitutes channel activity, additional EMRE subunits increase the [Ca ]c-threshold required for relief of gatekeeping. Endogenous channels most likely contain two EMRE subunits which tether two MICU1/2-dimers to the complex. These findings have important implications for developing new therapies to target diseases arising from dysregulation of mitochondrial Ca2+- transport. v TABLE OF CONTENTS DEDICATION ............................................................................................................................. III ACKNOWLEDGMENT .............................................................................................................. IV ABSTRACT ................................................................................................................................... V TABLE OF CONTENTS ............................................................................................................. VI LIST OF TABLES ..................................................................................................................... VIII LIST OF ILLUSTRATIONS ....................................................................................................... IX 2+ CHAPTER 1: INTRODUCTION TO MITOCHONDRIAL CA TRANSPORT ................. 1 2+ The physiological importance of mitochondrial Ca uptake .................................................... 1 2+ Regulation of TCA cycle enzymes by Ca ions .......................................................................... 1 2+ Physiological effects of mitochondrial Ca overload ................................................................... 3 2+ Molecular identity of the uniporter: the primary pathway for Ca across the IMM ................ 4 MCU: the pore-forming subunit of the uniporter .......................................................................... 5 MICU1, 2 and 3: Accessory proteins that regulate uniporter activity ........................................... 8 EMRE: A small transmembrane subunit with a dual role in MCU regulation ............................... 9 MCUR1: An assembly factor that regulates uniporter activity ................................................... 12 2+ Other mechanisms for Ca transport in mitochondria............................................................ 12 CHAPTER 2: REGULATION OF MCU CHANNEL ACTIVITY BY MICU1 AND 2 ........ 15 Materials and methods: ..............................................................................................................
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