University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-9-2020 Novel regulatory roles of endocytic membrane trafficking oteinspr in mitochondrial homeostasis Trey Farmer University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Farmer, Trey, "Novel regulatory roles of endocytic membrane trafficking oteinspr in mitochondrial homeostasis" (2020). Theses & Dissertations. 440. https://digitalcommons.unmc.edu/etd/440 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. i Novel regulatory roles of endocytic membrane trafficking proteins in mitochondrial homeostasis TITLE PAGE By Trey Farmer A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biochemistry & Molecular Biology Graduate Program Under the Supervision of Professor Steve Caplan University of Nebraska Medical Center Omaha, Nebraska May, 2020 Supervisory Committee: Paul Sorgen, Ph.D. Justin Mott, M.D., Ph.D. Keith Johnson, Ph.D. ii Novel regulatory roles of endocytic membrane trafficking proteins in mitochondrial homeostasis Trey Farmer, Ph.D. University of Nebraska, 2020 Supervisor: Steve Caplan, Ph.D. ABSTRACT Endocytic membrane trafficking is a basic cell process that is critical for regulating the transport of lipids and proteins. Our lab focuses on the cellular functions and mechanisms of the proteins that regulate these pathways. A key family of regulatory proteins is the C-terminal Eps15 Homology Domain (EHD) protein family. The EHD family includes EHD1-4, which are ubiquitously expressed in mammalian tissues. While these isoforms do have some overlapping functions, each protein also has distinct activities in regulating the shape and fission of membranes throughout the endocytic pathways. Specifically, EHD1 uses ATP hydrolysis to induce constriction and fission of endocytic membranes. EHD1 is recruited to tubular recycling endosomes (TREs) by interacting with Molecules Interacting with CAsL-Like 1 (MICAL-L1) and it performs fission to release cargo-containing vesicles from the TRE. Our lab demonstrated that upon EHD1 depletion, the TREs become elongated due to the lack of fission and the receptors that recycle through this pathway display impaired recycling to the plasma membrane. Furthermore, our lab and others have shown that EHD1 not only interacts with MICAL-L1, but also with a variety of other proteins, such as the retromer cargo selection complex (CSC), which is known to regulate the trafficking of membranes iii and proteins between endosomes and the Golgi complex. Recently, the proposed role of VPS35, a core protein of the retromer complex, has expanded, and it was found to interact with and control the mitochondrial fission protein, Drp1. However, the connection between EHD1 and the retromer and their role in mitochondrial homeostasis is less clear. It was previously thought that endocytic regulatory proteins exclusively impacted membrane trafficking pathways, but recent studies suggest that endocytic regulatory proteins play a role in many other pathways including ciliogenesis, centrosome disengagement, and mitochondrial homeostasis. Herein, I describe a novel role for in endocytic regulatory proteins in controlling mitochondrial fission and mitochondrial-induced apoptosis. My studies led to a model by which EHD1 regulates the localization of the retromer within the cell; accordingly, when EHD1 is absent, the retromer no longer regulates the mitochondrial fission protein, Drp1. In addition, I demonstrate for the first time, a connection between endocytic proteins and apoptosis by proposing a model for an expanded role for the retromer complex in regulating mitochondrial-induced apoptosis through the trafficking of the anti-apoptotic protein, Bcl-xL. iv TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii TABLE OF CONTENTS .............................................................................................................. iv TABLE OF FIGURES .................................................................................................................... x LIST OF ABBREVIATIONS ....................................................................................................... xii ACKNOWLEDGEMENTS ........................................................................................................ xix Chapter I ......................................................................................................................................... 1 1. ENDOCYTIC TRAFFICKING ............................................................................................. 2 1.1 Overview .......................................................................................................................... 2 1.2 Modes of internalization ................................................................................................ 3 1.2.1 Clathrin-mediated endocytosis (CME) ................................................................. 5 1.2.2 Clathrin-Independent Endocytosis (CIE) ............................................................. 7 1.2.3 Clathrin-Independent Carriers/GPI-AP-enriched early endosomal compartment (GLIC/GEEC) ............................................................................................ 9 1.2.4 Arf6 mediated pathway ........................................................................................ 10 1.2.5 CIE of interleukin-2 receptor ................................................................................ 11 1.3 Sorting at the Early Endosome (EE)/Sorting Endosome (SE) ................................. 11 v 1.4 Sorting cargos to the LE/lysosome for degradation ................................................. 13 1.5 Sorting cargos for recycling back to the PM .............................................................. 14 1.6 Sorted cargos destined for the trans-Golgi network (TGN) ................................... 16 2. REGULATORS OF ENDOCYTIC TRAFFICKING......................................................... 20 2.1 Overview ........................................................................................................................ 20 2.2. Rab GTPases and their effector proteins .................................................................. 21 2.3 v-SNARE and t-SNARE proteins ................................................................................ 27 2.4 C-terminal Eps15 homology domain (EHD) protein family .................................. 28 2.4.1 EHD1 ........................................................................................................................ 30 2.4.2 EHD2 ........................................................................................................................ 33 2.4.3 EHD3 ........................................................................................................................ 34 2.4.4 EHD4 ........................................................................................................................ 35 3. REGULATION OF MITOCHONDRIAL FUSION/FISSION ........................................ 35 3.1. Overview ....................................................................................................................... 35 3.2 Effectors of fusion ......................................................................................................... 37 3.2.1 Mitofusin-1 and Mitofusin-2 (Mfn1 and Mfn2) ................................................. 37 3.2.2 Optic atrophy protein 1 (OPA1) ........................................................................... 38 3.3 Effectors of fission ......................................................................................................... 40 3.3.1 ER/mitochondria contact sites .............................................................................. 40 vi 3.3.2 Dynamin-related protein 1 (Drp1) ....................................................................... 41 3.3.3 Dynamin-2 (Dyn2) ................................................................................................. 42 3.4 Other modes of regulating mitochondrial fusion and fission ................................ 44 3.4.1. Indirect role of VPS35 in mitochondrial fusion ................................................ 45 3.4.2. Indirect role of VPS35 in mitochondrial fission ................................................ 46 4. REGULATION OF MITOCHONDRIA INDUCED APOPTOSIS ................................ 47 4.1 Overview ........................................................................................................................ 47 4.2 Bcl-2 protein family regulation ................................................................................... 49 5. SUMMARY AND CONCLUSIONS ................................................................................