Glasgow Theses Service http://theses.gla.ac.uk/ theses@gla.ac.uk Cowan, Marianne (2014) A role for the endosomal SNARE complex and tethers in autophagy. PhD thesis. http://theses.gla.ac.uk/5046/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given A ROLE FOR THE ENDOSOMAL SNARE COMPLEX AND TETHERS IN AUTOPHAGY A thesis submitted to the INSTITUTE OF MOLECULAR, CELL AND SYSTEMS BIOLOGY For the degree of DOCTOR OF PHILOSOPHY by Marianne Cowan College of Medical, Veterinary and Life Sciences Institute of Molecular, Cell and Systems Biology University of Glasgow October 2013 2 Autophagy is a major route for lysosomal and vacuolar degradation in mammals and yeast respectively. It is involved in diverse physiological processes and implicated in numerous pathologies. The process of autophagy is initiated at the pre-autophagosomal structure and is characterised by the formation of a double membrane vesicle termed the autophagosome which sequesters cytosolic components and targets them for lysosomal/vacuolar degradation. The molecular mechanisms that regulate autophagosome formation are not fully understood. The conserved oligomeric Golgi (COG) complex is a hetero-octameric tethering factor implicated in autophagosome formation which interacts directly with the target membrane SNARE proteins Syntaxin 6 and Syntaxin 16 via the Cog6 and Cog4 subunits respectively. The work presented in this thesis demonstrates direct interaction of the yeast orthologue of Syntaxin 16, Tlg2, with Cog2 and Cog4. In addition, I investigated binding of the COG complex subunits to Tlg1, Vti1 and Snc2, the partner SNARE proteins of Tlg2. Direct interaction of Tlg1, the yeast orthologue of Syntaxin 6, with Cog1, Cog2 and Cog4 were observed. Given that Tlg2 has previously been shown to regulate autophagy in yeast, these data support a conserved role for the COG complex in mediating autophagosome formation through regulation of SNARE complex formation. In addition to investigating binding of COG complex subunits to the endosomal SNARE complex, I have also investigated a role for autophagy in regulating Tlg2 levels. The SM protein Vps45 has previously been shown to stabilise Tlg2 cellular levels. Our laboratory has demonstrated a role for both the proteasome and vacuole in the degradation of Tlg2. Here I demonstrated a role for autophagy in the regulation of Tlg2 levels and show that Swf1-mediated palmitoylation may serve to protect Tlg2 from being selectively targeted for autophagy. I also investigated the effects of the yeast T238N mutation on Vps45 function. The analogous mutation in human Vps45 has recently been associated with congenital neutropenia. Vps45 function is best characterised in yeast where it associates with membranes via Tlg2 and is required for membrane traffic from the trans-Golgi network into the endosomal system. Cellular levels of Vps45 T238N were destabilised and a concomitant reduction in Tlg2 levels was also observed. Vacuolar protein sorting remained unaffected in yeast cells harboring Vps45 Abstract 3 T238N but was subjected to increased apoptosis under hydrogen peroxide- mediated stress. This identifies a novel role for Vps45 in maintaining cell viability. Finally, I also investigated a role for endosomal trafficking and autophagy in C.elegans post-embryonic development and identified a role for these pathways in the clearance of the pre-moult increase in intracellular membranes and cuticular formation. 4 Table of Contents Abstract 2 List of Tables 8 List of Figures 9 Acknowledgements 13 Author’s Declaration 14 Definitions/Abbreviations 15 Chapter 1 – Introduction 18 1.1 Autophagy 19 1.1.1 Identification of autophagy 19 1.1.2 Functional significance of autophagy 20 1.1.3 Autophagy versus the cytosol-to-vacuole targeting pathway 21 1.1.4 The process of autophagy 22 1.1.5 Ubiquitination and selective autophagy 28 1.1.6 Regulation of autophagy by signalling pathways 30 1.1.7 Autophagy in disease and development 31 1.2 SNARE proteins 32 1.2.1 Structure and function of SNARE proteins 32 1.2.2 Expression and localisation of SNARE proteins 34 1.2.3 The endosomal SNARE complex 34 1.2.4 Syntaxin 16 is the mammalian orthologue of Tlg2 35 1.2.5 Regulation of Tlg2 cellular levels 36 1.2.5.1 Protein palmitoylation 37 1.3 The SM family of proteins 38 1.3.1 SM protein structure 38 1.3.2 Regulation of membrane fusion by SM proteins 39 1.3.3 Other SM protein interactions 40 1.3.4 Identification of the SM protein Vps45 41 1.4 Tethering proteins 42 1.4.1 Function of the COG tethering complex 43 1.4.2 Molecular structure of the COG complex 44 1.5 C.elegans: An introduction 45 1.5.1 C.elegans post-embryonic development 46 1.5.2 C.elegans cuticle 47 1.5.3 Temporal expression of cuticle collagen genes 48 5 1.5.4 Collagen protein structure 48 1.5.5 UNC-51 is the C.elegans ortholog of yeast Atg1 49 1.5.6 VPS-45 function in C.elegans 50 1.6 Project aims 51 Chapter 2 – Materials and Methods 53 2.1 Materials 53 2.1.1 Antibodies 54 2.1.2 Bacterial, yeast and nematode strains 55 2.1.3 Growth media 57 2.2 Molecular Biology 58 2.2.1 Purification of plasmid DNA from E.coli 58 2.2.2 Agarose gel electrophoresis 61 2.2.3 Gel extraction and purification of DNA 62 2.2.4 Polymerase Chain Reaction 62 2.2.5 Site-directed mutagenesis 65 2.2.6 Restriction endonuclease digestion of DNA 66 2.2.7 Ligation of DNA 67 2.3 Protein analysis 68 2.3.1 SDS-polyacrylamide gel electrophoresis 68 2.3.2 Coomassie™ blue staining 68 2.3.3 Western blot transfer 69 2.3.4 Immunological detection of proteins 69 2.4 IgG affinity purification 70 2.5 General yeast methods 71 2.5.1 Cryopreservation and maintenance of yeast cell stock 71 2.5.2 Preparation of competent yeast cells 71 2.5.3 Transformation of competent yeast cells 72 2.5.4 Preparation of yeast whole cell lysates 72 2.5.4.1 Rapid Twirl buffer lysis procedure 73 2.5.4.2 Glass bead lysis procedure 73 2.5.5 Isolation of yeast genomic DNA 74 2.6 Production of mutant yeast strains by homologous recombination 75 2.7 Carboxypeptidase Y overlay assay 76 2.8 Palmitoylation assays 77 2.8.1 Hydroxylamine treatment 77 2.8.2 Acyl resin-assisted capture 78 2.9 Bradford protein assay 80 2.10 Hydrogen peroxide halo assay 81 2.11 Purification of recombinant fusion proteins from E.coli 81 6 2.11.1 Preparation of competent bacterial cells 81 2.11.2 Transformation of competent bacterial cells 82 2.11.3 Cryopreservation and maintenance of plasmid DNA 82 2.11.4 Expression of recombinant fusion proteins 82 2.11.5 Purification of GST fusion proteins 84 2.11.6 Purification of Protein A fusion proteins 85 2.12 Protein interaction assays 86 2.12.1 GST and Protein A pull-down assays 86 2.12.2 Yeast two-hybrid assay 87 2.13 C.elegans methods 89 2.13.1 Maintenance of C.elegans in culture 89 2.13.2 Preparation of E.coli OP50-1 liquid culture 89 2.13.3 Cryopreservation and recovery of C.elegans 90 2.13.4 Isolation of C.elegans genomic DNA 90 2.13.5 Preparation of C.elegans whole animal lysates 91 2.13.6 C.elegans genetic crosses 91 2.13.7 Nomarski microscopy 91 2.13.8 Immunofluorescence of C.elegans 92 Chapter 3 – Endosomal SNAREs and autophagy 93 3.1 Overview and aims 93 3.2 Results 94 3.2.1 Yeast two-hybrid assays 94 3.2.1.1 Summary of yeast two-hybrid interactions 109 3.2.2 Pull-down assays 110 3.2.2.1 Expression and purification of recombinant fusion proteins 110 3.2.2.2 Detection of chromosomally expressed HA-tagged Cog proteins 116 3.2.2.3 Tlg2 directly associates with COG complex subunits 117 3.2.2.4 Tlg1 directly associates with Cog1 122 3.2.2.5 Functional significance of the Tlg1 and Cog1 interaction 123 3.2.2.6 Tlg1 directly associates with Cog2 and Cog4 125 3.2.2.7 Summary of pull-down interactions 128 3.3 Chapter summary 129 Chapter 4 – Regulation of Tlg2 steady-state levels 131 4.1 Overview and aims 131 4.2 Results 132 4.2.1 Vps45 regulates Tlg2 steady-state protein levels 132 4.2.2 Tlg2 steady-state protein levels are regulated by the vacuole 133 4.2.3 Tlg2 is regulated in an autophagy-dependent manner 134 7 4.2.4 A role for palmitoylation in the regulation of Tlg2 141 4.3 Chapter summary 148 Chapter 5 – The T238N mutation in yeast Vps45 150 5.1 Overview and aims 150 5.2 Results 151 5.2.1 Generation of the Vps45 T238N mutation in yeast 151 5.2.2 The yeast Vps45 T238N position localises to domain 3a 153 5.2.3 Tlg2 is destabilised by the Vps45 T238N mutation in yeast 154 5.2.4 CPY is correctly sorted in yeast harboring the Vps45T238N mutation 156 5.2.5 The T238N mutation in yeast VPS45 leads to increased apoptosis 158 5.2.6 Chapter summary 162 Chapter 6 – Autophagy and endosomal trafficking in C.elegans development 164 6.1 Overview and aims 164 6.2 Results 165 6.2.1 Disruption of autophagy in dpy-10 mutant backgrounds 167 6.2.2 Disruption of endosomal trafficking in dpy-10 mutant backgrounds 170 6.2.3 Characterisation of C.elegans strains 175 6.2.4 C.elegans development and a role for autophagy 178 6.2.4.1 Morphological characterisation of autophagy deficient C.elegans 179 6.2.4.2 Cuticular localisation of DPY-7 in autophagy deficient C.elegans 182 6.2.5 C.elegans development and a role for endosomal trafficking 184 6.2.5.1 Cuticular localisation of DPY-7 in endosomal trafficking deficient C.elegans 184 6.2.5.2 Monitoring soluble DPY-7 in endosomal trafficking deficient C.elegans 185 6.3 Chapter summary 189 Chapter 7 – Discussion 190 7.1 Endosomal SNAREs and autophagy 190 7.2 Regulation of Tlg2 steady-state levels 194 7.3 The T238N mutation in yeast Vps45 195 7.4 Autophagy and endosomal trafficking in C.elegans development 197 References 200 Publications 219 8 List of Tables Table 2-1 Antibiotics used in this study 53 Table 2-2 Antibodies used in this study 54 Table 2-3 E.coli strains used in this study 55 Table 2-4 S.cerevisiae strains used in this study 56 Table 2-5 C.elegans strains used in this study 57 Table 2-6 List of plasmids used in this study 59 Table 2-7 Oligonucleotides used in this study 63 Table 2-8 Standard PCR reaction mix 64 Table 2-9 Standard PCR conditions 64 Table 2-10 SDM PCR conditions 65 Table 2-11 Standard restriction enzyme digest 66 Table 2-12 DNA ligation reaction 67 Table 3-1 Summary of yeast two-hybrid interactions 109 Table 3-2 Summary of pull-down interactions 128 9 Figure 1-1. The process of autophagy 18 Figure 1-2. Schematic representation of the endosomal system, autophagy and the Cvt pathway in yeast. 26 Figure 1-3. Schematic overview of ubiquitination 29 Figure 1-4. Regulation of autophagy by TORC1 30 Figure 1-5. Domain structure of the syntaxin proteins 32 Figure 1-6. Closed and open conformations of the SNARE proteins 33 Figure 1-7. Transmembrane domain protein sequence alignment of yeast SNARE proteins 38 Figure 1-8. Modes of SM protein binding to SNARE proteins 40 Figure 1-9. Schematic diagram of membrane fusion 44 Figure 1-10. Architecture of the COG complex 45 Figure 1-11. C.elegans development 46 Figure 1-12. Structural organisation of the C.elegans cuticle 47 Figure 2-1. One-step gene replacement primers 75 Figure 2-2. One-step gene replacement by homologous recombination 76 Figure 2-3. Summary flow chart of hydroxylamine treatment protocol 78 Figure 2-4. Recombinant fusion protein expression summarised 83 Figure 2-5. Summary flow chart of yeast two-hybrid protocol 88 Figure 3-1. Yeast two-hybrid schematic 95 Figure 3-2. Yeast two-hybrid plasmids 96 Figure 3-3. Yeast two-hybrid interactions between AD-Tlg2 cyto and BD Cog constructs 99 Figure 3-4. Yeast two-hybrid interactions between AD Tlg2 cyto ∆N36 and BD Cog constructs 100 Figure 3-5. Yeast two-hybrid interactions between AD-Tlg2 cyto ∆Habc and BD Cog constructs 101 Figure 3-6. Yeast two-hybrid positive and negative interaction controls for BD Cog constructs 102 Figure 3-7. Expression of the yeast two-hybrid AD-Tlg2 cyto , AD-Tlg2 cyto ∆N36 and AD-Tlg2 cyto ∆Habc fusion proteins 103 Figure 3-8. Yeast two-hybrid interactions between BD-Tlg2 cyto and AD Cog constructs 105 List of Figures [...]... this observed increase in autophagy to be glucagon-mediated 1.1.2 Functional significance of autophagy Autophagy is an evolutionary conserved and adaptive catabolic process that plays a central role in maintaining intracellular homeostasis and thereby cellular health The term autophagy directly translates to ‘self-eating’ and it is a major route for lysosomal/vacuolar degradation in eukaryotes (Reggiori... simultaneously bind ubiquitin and the autophagosomeassociated ubiquitin-like proteins Atg8 and LC3, respectively, provided insight into how protein cargo can be selectively targeted to the vacuole and lysosome via autophagy (Pankiv et al., 2007; Noda et al., 2008) The ubiquitin binding protein p62 contains a N-terminal LC3-interacting region (LIR) and a carboxy (C)-terminal ubiquitin-associated (UBA) domain (Pankiv... encodes the vacuolar alkaline phosphatase which contains an N-terminal transmembrane domain Pho8 is delivered to the vacuole via the secretory pathway and its transmembrane domain signals translocation into the ER Pho8∆60 lacks the transmembrane domain and instead localises to the cytosol Pho8∆60 is exclusively delivered to the vacuole via autophagy thus Pho8∆60 activity can be utilised to quantify the magnitude... for binding to Atg1 Upon binding, dephosphorylated Atg13 activates Atg1 kinase activity (Kijanska et al., 2010) Association between Atg1 and Atg13 is required for the initiation of autophagy (Kamada et al., 2000) These events implicate an important regulatory role for TORC1 kinase activity in autophagy Figure 1-4 Regulation of autophagy by TORC1 Target of rapamycin complex 1 (TORC1) activity negatively... et al., 2010) Localization of the COG complex subunits to the PAS combined with its role as a tethering factor implicates a role for this complex during the early phases of autophagy More recent evidence support a role for SNARE- mediated homotypic fusion reactions in the formation of autophagosomes and thereby autophagy Atg16 forms a complex 26 Marianne Cowan, 2013 Chapter 1 - Introduction with the Atg12-Atg5... squares) The SNARE domain is the defining feature of syntaxin proteins (represented by a diagonally striped rectangle) and is followed by the C-terminal transmembrane domain (TMD; represented by a solid dark grey rectangle) Adapted from (Fernandez et al., 1998) In addition to the SNARE domain, the syntaxin SNARE proteins possess an autonomously folded amino (N)-terminal domain that forms a three-helix... sequester and deliver specific enzymes, such as aminopeptidase I (Klionsky et al., 1992) and αmannosidase (Yoshihisa & Anraku, 1990), from the cytosol to the vacuole; in contrast, autophagy is an inducible degradative pathway that terminates in the lysosomal/vacuolar compartment (Yang & Klionsky, 2010) Transport vesicle formation is a key regulatory step of the Cvt and autophagic pathways and the preautophagosomal... autophagy and the Cvt pathway is depicted in Figure 1-2 Localisation of the key proteins under investigation in the current study, Tlg2, Vps45 and the COG complex, are indicated (Figure 1-2) 25 Marianne Cowan, 2013 Chapter 1 - Introduction Figure 1-2 Schematic representation of the endosomal system, autophagy and the Cvt pathway in yeast Key trafficking pathways within the endosomal system, autophagy. .. Atg17 is unaffected in core atg mutant strains; in contrast, the PAS localisation of the remaining core Atg proteins is impaired in atg17 (Suzuki et al., 2007) The PAS localisation of the Atg17-Atg29-Atg31 complex and its subsequent binding to Atg11 via Atg17 (Yorimitsu & Klionsky, 200 5a) is regulated by phosphorylation of Atg29 (Mao et al., 2013) Binding between Atg11 and the Atg17-Atg29-Atg31 complex. .. ubiquitin-like conjugation systems and the integral membrane protein Atg9 (Noda et al., 2000) In addition to these core Atg proteins, autophagy- and Cvt-specific proteins have also been identified (Kawamata et al., 2008) Despite sharing similar morphological features, important differences exist between autophagy and the Cvt pathway The Cvt pathway is a constitutively active biosynthetic pathway that serves . identifies a novel role for Vps45 in maintaining cell viability. Finally, I also investigated a role for endosomal trafficking and autophagy in C.elegans post-embryonic development and identified a role. regulate autophagy in yeast, these data support a conserved role for the COG complex in mediating autophagosome formation through regulation of SNARE complex formation. In addition to investigating. resin-assisted capture AD activation domain APS ammonium persulphate ATG autophagy related gene ATP adenosine triphosphate BD binding domain bp base pairs BSA bovine serum albumin CaCl 2 calcium