Thesis
Reference
The exocyst complex in "Dictyostelium discoideum" : localisation and functional characterisation
ESSID, Miriam
Abstract
L'objectif de cette étude était de disséquer le trafic membranaire chez "Dictyostelium." Nous avons caractérisé la fonction de la petite GTPase Rab8a et de son effecteur potentiel, l'exocyste pendant l'expulsion de liquide par la vacuole contractile (VC). La VC est un système tubulaire et vésiculaire qui contrôle l'équilibre osmotique de "Dictyostelium." La mutation de Rab8a, Drainin ou LvsA affectent la morphologie de la VC et la localisation de l'exocyste. Rab8a-GTP recrute l'exocyste à la membrane de la VC et favorise l'attachement de la VC avec la membrane plasmique afin de permettre la formation d'un pore et l'expulsion de liquide. L'impossiblité de supprimer "sec15", une sous-unité de l'exocyste, indique que l'exocyste est essentiel pour la viabilité cellulaire. Effectivement, la recherche des partenaires d'interaction de l'exocyste a révélé d'autres rôles potentiels dans le trafic vésiculaire, la phagocytose, l'exocytose et mobilité cellulaire
ESSID, Miriam. The exocyst complex in "Dictyostelium discoideum" : localisation and functional characterisation. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4155
URN : urn:nbn:ch:unige-51603
DOI : 10.13097/archive-ouverte/unige:5160
Available at:
http://archive-ouverte.unige.ch/unige:5160
Disclaimer: layout of this document may differ from the published version.
Département de biochimie Dr Thierry Soldati ___________________________________________________________________________
The Exocyst Complex in Dictyostelium discoideum:
Localisation and Functional Characterisation
THÈSE
présentée à la Faculté des sciences de l'Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie
N° 04.347.050
par Miriam Essid
de
Guxhagen (Allemagne)
GENÈVE ReproMail
2009
List of Content
page
0 Summary 1
0.1 Summary (english) 1
0.2 Résumé 3
1. Introduction 5
1.1 Membrane trafficking 5
1.1.1 The secretory pathway 7
1.1.2 The endosomal pathway 7
1.2 Rab proteins, a member of the superfamily of small GTPases 8
1.3 Tethering complexes 10
1.3.1 The COG complex 12
1.3.2 The GARP complex 12
1.3.3 The HOPS complex 12
1.3.4 The CORVET complex 12
1.3.5 The TRAPPI / II complexes 13
1.3.6 The Dsl1 complex 13
1.3.7 The exocyst complex 13
1.3.7.1 Structure of the exocyst complex 14
1.3.7.2 Assembly and targeting of the exocyst complex 15
1.3.7.3 Targeting of the exocyst complex to sites of exocytosis 15
1.3.7.4 The exocyst in focal exocytosis 18
1.3.7.4.1 Exocyst-mediated vesicle trafficking in polarised epithelial cells 18 1.3.7.4.2 Exocyst-mediated transport of Glut4 vesicles to the PM 19 1.3.7.4.3 Exocyst-mediated vesicle delivery during cytokinesis 19 1.3.7.4.4 Exocyst-mediated neurit outgrowth and synaptogenesis 20
1.3.7.4.5 Exocyst-mediated invadopodia formation 20
1.3.7.5 The exocyst in motility 20
1.4 The model organism Dictyostelium discoideum 21
1.4.1 Membrane trafficking in Dictyostelium 21
1.4.1.1 The contractile vacuole 23
1.4.2 Rab proteins in Dictyostelium 25
1.4.3 Drainin 26
1.4.4 LvsA 27
1.4.5 Tethering complexes in Dictyostelium 27
1.5 Aim of the thesis 29
2. Material and Methods 30
2.1 Material 30
2.1.1 Antibodies 30
page
2.1.2 Media, Buffers and Solutions 30
2.1.2.1 Media 30
2.1.2.2 Buffers and Solutions 31
2.1.3 Antibodies 32
2.1.4 Molecular weight markers 35
2.1.5 Kits 35
2.1.6 Cell lines 35
2.1.7 Vectors and plasmids 35
2.1.8 Primer 37
2.1.9 Dictyostelium cell lines generated during this work 37
2.2 Methods 38
2.2.1 Cell culture 38
2.2.1.1 Long term storage of cells 38
2.2.1.1.1 Cell freezing 38
2.2.1.1.2 Spore freezing 38
2.2.2 Electroporation of Dictyostelium 38
2.2.3 Isolation of genomic DNA from Dictyostelium 39
2.2.3.1 Preparation of chromosomal DNA from Dictyostelium grown in 12-well plates 41
2.2.4 Preparation of electroporation-competent E. coli 42
2.2.5 Transformation of E. coli cells 43
2.2.6 Cloning 43
2.2.7 Polymerase Chain Reaction (PCR) 43
2.2.8 Reverse Transcriptase-PCR (RT-PCR) with Roche Titan One Tube RT-PCR System 44
2.2.9 Ligation 45
2.2.10 Colony PCR 46
2.2.11 DNA precipitation 46
2.2.12 Separation of nucleic acids on an agarose gel 46
2.2.13 Nucleic acid quantification 46
2.2.14 Digestion of DNA with restriction endonucleases 47
2.2.15 Cloning strategies 47
2.2.15.1 Cloning into vectors for protein fusion to various tags 47 2.2.15.2 Cloning strategy for "classical" knock-out of Sec15 48 2.2.15.3 Coning strategy for tetracycline-regulatable gene knock-down 50 2.2.15.4 Cloning strategy for DD-domain regulatable expression of Rab8a 51
2.2.16 Biochemical Methods 53
2.2.16.1 Cell lysates 53
2.2.16.2 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 53
2.2.16.3 Western blots 53
2.2.16.4 Immunodetection 53
2.2.16.5 Staining of SDS-PAGE with Coomassie 53
2.2.16.6 Silver staining 54
page 2.2.16.7 GFP co-immunoprecipitation with Dictyostelium cell lysate 55 2.2.16.7.1 GFP co-immunoprecipitation with Dictyostelium cell lysate 55 2.2.16.7.2 anti-GFP co-immunoprecipitation with Dictyostelium cell lysate using an
antibody directly coupled to beads 56
2.2.16.8 Purification of recombinant GST-tagged proteins expressed in E. coli 57
2.2.16.9 Mass-spectrometry and protein identification 59
2.2.16.10 Fixation of cells on coverslips and immunofluorescence 59
2.2.16.11 Electron microscopy 60
3. Results 61
3.1 The small GTPase Rab8a localises to the contractile vacuole 61 3.2 Mutations of Rab8a affect the morphology and discharge behaviour of
the contractile vacuole 62
3.2.1 Mutation of Rab8a changes its cellular localisation 62
3.2.2 The morphology of the CV is dramatically altered in DD-GFP-Rab8a
CA and DD-GFP-Rab8a DN cells 64
3.2.3 CV bladder diameter and number of CV bladders is increased in
DD-GFP-Rab8a DN cells 68
3.3 The exocyst complex, a potential effector of Rab8a 70
3.3.1 The Dictyostelium exocyst shares high homology with the mammalian exocyst 70
3.3.2 The exocyst localises to the contractile vacuole 71
3.3.3 Rab8a recruits the exocyst to the contractile vacuole 74
3.4 Knock-down of the exocyst subunit Sec15 75
3.4.1 Diameter of CV bladders in Sec15 knock-down cells is elevated in cells
with three or four CV bladders 77
3.5 Screen for interaction partners of the exocyst 80
3.5.1 Yeast 2-hybrid screen with full-length Sec15 and the C-terminal half of Exo70 80
3.5.2 Anti-GFP co-IPs and mass-spectrometry analysis 83
3.5.3 Drainin, an interaction partner of the exocyst 90
3.5.3 LvsA, an interaction partner of the exocyst 93
3.6 Other potential functions of the exocyst 96
3.6.1 The exocyst in phagocytosis 96
3.6.2 The exocyst in exocytosis of post lysosomes 97
3.6.3 The exocyst in streaming cells 98
4. Discussion 100
4.1 Rab8a and the exocyst regulate contractile vacuole bladder discharge 100 4.2 New interaction partners connecting to new functions for the Dictyostelium
exocyst 103
5. Supplementary data 106
page 5.1 Alignments of Dictyostelium exocyst proteins sequences with
human and yeast (Saccharomyces cerevisiae) exocyst protein sequences 106
5.1.1. Sec3 106
5.1.2 Sec5 108
5.1.3 Sec6 109
5.1.4 Sec8 111
5.1.5 Sec10 113
5.1.6 Sec15 114
5.1.7 Exo70 116
5.1.8 Exo84 118
5.2 Rab proteins in Dictyostelium 119
5.2.1 Rab4 119
5.2.2 Rab11a 121
5.2.3 Rab11b 123
5.2.4 Rab11c 124
5.2.5 Rab14 126
5.3 DNA sequences of exocyst subunits 127
5.3.1 Sec3 127
5.3.2 Sec8 128
5.3.3 Sec15 130
5.3.4 Exo70 132
5.4 cDNA sequence of Rab proteins 133
5.4.1 Rab4 133
5.4.2 Rab8a 133
5.4.3 Rab11a 133
5.4.4 Rab11b 134
5.4.5 Rab11c 134
6. Bibliography 135
Acknowledgements 145
0.1 Summary (english)
The overall aim of this study was a better understanding of membrane trafficking in Dictyostelium, a model for animal cells. Therefore, we characterised the function of the small GTPase Rab8a and its potential effector the exocyst during contractile vacuole (CV) bladder discharge.
The small GTPase Rab8a regulates CV bladder discharge. The CV system controls the osmotic equilibrium of the Dictyostelium cells. Mutations of Rab8a affect CV discharge and lead to aberrant morphologies of the contractile vacuole (CV) system. Interestingly, this affects as well the localisation of potential Rab8a effector, the exocyst complex. We concluded that Rab8a-GTP recruits the exocyst to the CV bladder membrane for subsequent tethering of the CV bladder to the PM and pore formation. The exocyst is an octameric tethering complex functioning in cellular processes such as focal exocytosis, cytokinesis and cell migration. In Dictyostelium, the exocyst localises at the CV bladder. Knock-down of Sec15, an exocyst subunit shows only a very slight increase in CV bladder diameter as long as the cells have only one CV bladder per cell. This changes completely in cells with three or four CV bladders, indicating that lack of Sec15 decreases the efficiency of pore formation.
To investigate further the role of the exocyst in CV discharge, we monitored the exocyst in mutants impaired in CV function, like Drainin and LvsA. Drainin, a Rab-GAP-like protein, is proposed to be necessary for pore formation. Drainin-null cells show enlarged CV bladders (Becker et al., 1999). In these cells, GFP-Sec15 strongly accumulates at the contact site between CV and PM. LvsA is a BEACH-domain protein of the Shediak Higashi family (Gerald et al., 2002). In lvsA-null cells the CV bladder discharges, but stays collapsed beneath the PM. GFP-Sec15 and Rab8a localise at the sites of collapsed CV bladder.
Taken together, this results in the following model. Under hypoosmotic stress, Rab8a, the exocyst and Disgorgin, a Rab8a-GAP are recruited to the CV bladder (Du et al., 2008). At the PM, Disgorgin activity is enhanced by Drainin and starts to catalyse the hydrolysis of Rab8a-GTP, which leads Rab8a-GTP and exocyst concentration in the CV bladder - PM contact zones. Tethering of the CV bladder to the PM, mediated by the exocyst, is followed by pore formation and discharge of the CV bladder. After, the CV bladder de-tethers from the PM and re-integrates into the CV system to start a new cycle of refill and discharge. We propose that de-tethering of the CV bladder from the PM depends on hydrolysis of Rab8a-GTP and a not yet identified action of LvsA.
The second branch of the project concentrated on the identification of other potential exocyst functions. The failure in Sec15 knock-out lead us assume that Sec15 is an essential gene. Therefore, the exocyst must function in other processes than CV discharge, which are essential for cell viability.
To identify these functions, we searched for interaction partners of the exocyst by yeast 2-Hybrid screens and co-immunoprecipitations.
We identified proteins like, subunits of COPI, the AP1 subunit B1 and Huntingtin, which indicate a role for the exocyst in vesicle transport.
We found proteins like Comitin (Schreiner et al., 2002), the subunit 4 of the Arp2/3 complex, ArcD and dynamin (Insall et al., 2001; Wienke et al., 1999) that are all found in the phagocytic cup, suggesting a function of the exocyst in phagocytosis.
We identified UbqC and several subunits of the proteasome, indicating that the exocyst, including Rab8a and Disgorgin might be targets for ubiquitination and final proteasomal degradation.
Additionally, we wanted to know more about the regulation of the exocyst by small GTPases. We identified the small GTPase Rac1a. Based on the different phenotypes of Rac1a knock-out cells (Dumontier et al., 2000), we propose that Rac1a is important for exocyst assembly at the PM. It might have a Cdc42-like targeting role in Dictyostelium (Adamo et al., 2001; Zhang et al., 2001).
In conclusion, we demonstrate that the small GTPase Rab8a recruits the tethering factor exocyst to the CV bladder prior to discharge. Rab8a and the exocyst are important for pore formation and CV discharge. Mutation of either one results in delay of discharge and morphological changes of the CV system. Furthermore we unravel other potential functions of the exocyst, like a role in vesicle trafficking, phagocytosis or exocytosis. Targeting of the exocyst to the PM might be regulated by the small GTPase Rac1a.
0.2 Résumé
L’objectif général de cette étude a été de mieux comprendre le trafic membranaire chez Dictyostelium, qui est un modèle pour l’étude des cellules animales. Pour cela, nous avons caractérisé la fonction de la petite GTPase Rab8a et de son effecteur potentiel, l’exocyste, pendant l’expulsion de liquide par la vacuole contractile (VC).
La petite GTPase Rab8a régule l'expulsion par la VC. La VC est un système tubulaire et vésiculaire qui contrôle l'équilibre osmotique de Dictyostelium. Des mutations de la protéine Rab8a affectent l’expulsion par la VC et sont responsables de morphologies aberrantes pour ce système. Phénomène plus intéressant, cela affecte également la localisation de l’effecteur potentiel de Rab8a, l’exocyste.
Nous en avons conclu que Rab8a-GTP recrute l’exocyste à la membrane de la VC pour pouvoir ensuite lier celle-ci à la membrane plasmique et ainsi permettre la formation d’un pore. L’exocyste est un complexe d’attachement composé de 8 sous-unités. Il est impliqué dans des processus cellulaires tels que l’exocytose focalisée, la cytokinèse et la migration des cellules. Chez Dictyostelium, l’exocyste est localisé au niveau de la vacuole de la VC. Le "knock-down" de la protéine Sec15, une sous-unité de l'exocyste, ne provoque qu’une légère augmentation du diamètre de la vacuole de la VC, à condition que la cellule n’en contienne qu’une. Mais l’effet est d’autant plus important lorsque la cellule contient 3 ou 4 vacuoles de VC, indiquant que le manque de Sec15 diminue l'efficacité de la formation du pore.
Pour déterminer de façon plus précise le rôle de l'exocyste dans l'expulsion de la VC, nous avons observé l’exocyste chez différents mutants déficients pour cette fonction, comme la Drainin et la LvsA.
La Drainin, une protéine semblable à une Rab-GAP, est soupçonnée d’être nécessaire à la formation du pore. Les cellules drainin-nulles montrent en effet des vacuoles de la VC élargies (Becker et al., 1999). Dans ces cellules, GFP-Sec15 est fortement concentrée dans la zone de contact entre la VC et la membrane plasmique. LvsA est une protéine de la famille de celle impliquée dans le syndrome Shediak Higashi contenant un domaine BEACH (Gerald et al., 2002). Dans les cellules lvsA-nulles, les vésicules de VC sont capables d’expulser le liquide qu’elles contiennent. Cependant, après expulsion, les vacuoles de VC collapsent et restent accolées à la membrane plasmique. GFP-Sec15 et Rab8a se localisent au niveau de la vacuole de la VC collapsée. Tous ces résultats permettent d’aboutir au modèle suivant : sous stress hypo-osmotique Rab8a, l’exocyste et la disgorgin, une GAP pour Rab8a, sont assemblés au niveau de la vacuole de la VC (Du et al., 2008). La vacuole de la VC s'approche de la membrane plasmique, où l'activité de la disgorgin est augmentée par la drainin. Elle catalyse alors l'hydrolyse de GTP par Rab8a. Rab8a-GTP et l’exocyste sont ainsi enrichis au niveau de la zone de contact entre la vacuole de la VC et la membrane plasmique. L’attachement de la vacuole de la VC à la membrane plasmique via l’exocyste est suivi par la formation d’un pore et l’expulsion du contenu de la VC. La vacuole de la VC se détache ensuite de la membrane plasmique. Elle réintègre alors le système vésiculo-tubulaire de la VC et peut ainsi commencer un nouveau cycle de pompage et d’expulsion. Nous établissons l'hypothèse que le détachement de la vacuole de la VC de la membrane plasmique est dépendant de l’hydrolyse du GTP par Rab8a et d'une action pour l’instant inconnue de LvsA. La seconde partie du projet a consisté à identifier d’autres fonctions potentielles de l'exocyste.
L’échec à obtenir un "knock-out" de la protéine Sec15 nous a amené à en déduire que Sec15 est un gène essentiel. Nous en avons par conséquent conclu que l’exocyste doit avoir d’autres rôles que
l’expulsion de liquide par la VC, essentielle pour la viabilité cellulaire. Pour identifier ses fonctions, nous avons recherché des partenaires interagissant avec l'exocyste à l’aide d’un crible double-hybride dans la levure et de co-immunoprécipitations. Nous avons identifié différentes protéines, comme des sous-unités de COPI, la sous-unité B1 d’AP1 et Huntingtin, mettant potentiellement en évidence le rôle de l'exocyste dans le transport vésiculaire. Nous avons aussi trouvé des protéines comme la comitin (Schreiner et al., 2002), la sous-unité 4 du complexe Arp2/3, ArcD et la dynamine (Insall et al., 2001; Wienke et al., 1999), toute présentes au niveau de la coupe phagocytaire et suggérant donc un rôle de l’exocyste dans la phagocytose. De plus, nous avons montré que la protéine Sec15 est localisée avec la vacuoline et p80 au niveau des post-lysosomes, mettant à nouveau en évidence un possible rôle de l'exocyste dans l'exocytose. Nous avons aussi identifié UbqC et plusieurs sous-unités du protéasome, indiquant que l’exocyste ainsi que Rab8a et la disgorgine pourraient être des cibles de ubiquitination avant d’être finalement dégradés par le protéasome. De surcroît, nous avons également voulu en savoir plus sur la régulation de l'exocyste par des petites GTPase. Nous avons identifié la petite GTPase Rac1a. D’après les différents phénotypes obtenus après le knock-out de la protéine Rac1a (Dumontier et al., 2000), nous émettons l'idée que Rac1a est importante pour l'assemblage de l'exocyste à la membrane plasmique. Elle pourrait jouer chez Dictyostelium un rôle similaire à Cdc42 chez la levure (Adamo et al., 2001; Zhang et al., 2001).
Pour conclure, nous avons démontré que la petite GTPase Rab8a recrute le facteur d’attachement exocyste au niveau de la vacuole de la VC. Rab8a et l’exocyste sont importants pour la formation du pore et ensuite pour l’expulsion par la VC. Une mutation de l’un des deux provoque un retard dans l’expulsion du contenu de la VC et un changement dans la morphologie de ce système tubulo- vésiculaire. De plus, nous avons révélé d’autres rôles potentiels pour l’exocyste, comme un rôle dans le trafic vésiculaire, la phagocytose ou l’exocytose. L'attraction de façon ciblée de l’exocyste à membrane plasmique pourrait être régulée par la petite GTPase Rac1a.
1. Introduction
1.1 Membrane trafficking
In contrast to prokaryotic cells, a eukaryotic cell is divided in different compartments. These compartments are the nucleus, mitochondria and the organelles, which represent each a separate compartment. To ensure communication between the organelles the cell developed a highly dynamic system of membrane trafficking that depends mostly on vesicles moving from a donor to an acceptor compartment. To ensure an equilibrium, all steps of this process have to be carefully regulated, starting with vesicle budding, transport, tethering and subsequent fusion.
Key regulators of membrane trafficking are Rab GTPases. The first indication, for these nowadays well proven statements came from yeast. Mutation of Sec4p (a homolog to the mammalian Rab8) resulted in accumulation of trans golgi network (TGN) derived secretory vesicles (Salminen and Novick, 1987). The mechanism of action and function of the Rab proteins will be explained under 1.2
"Rab proteins, a member of the superfamily of small GTPases".
Vesicle and organelle movement is based on molecular motors that move along the cytoskeleton, which consists of two types of cytoskeletal filaments, microtubules and actin. Microtubules are important for long the long-range transport. They radiate from the microtubule organising centre (MTOC) close to the nucleus towards the plasma membrane (PM). In between the microtubules and in the cell periphery one finds a meshwork of actin filaments to ensure the transport over small distances.
Both types of cytoskeletal filament carry distinct molecular motors. Microtubule-dependent transport is mediated by kinesin and dynein. Kinesin is a plus end directed motor that moves from the cell centre towards the cell periphery. Dynein is a minus end directed motor that moves form the cell periphery towards the cell centre, the MTOC. Transport on actin filaments depends on myosins (Soldati and Schliwa, 2006).
When vesicles reach their destination, donnor and accpetor membranes fuse. This is mediated by tethering complexes and subsequently SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) interactions. Membrane tethering and fusion is of special interest in this thesis, therefore the role of tethering complexes, specially the exocyst complex, is separately discussed in chapter 1.3 "Tethering complexes". SNAREs are C-terminally anchored integral membrane proteins oriented towards the cytosol. The SNARE bundle bridges the two membranes, and its formation is thought to overcome the energy barrier preventing the two membranes from fusing. A v-SNARE on the vesicle and the cognate t-SNAREs on the target membrane assemble by formation of a 4-α-helix bundle. One helix is contributed by the v-SNARE, and three by t-SNAREs on the target membrane.
(Lupashin and Sztul, 2005).
Coming back from the details of vesicle fusion to membrane trafficking in general. We will have a closer look at membrane trafficking in mammalian cells. There are two main routes of membrane trafficking, the secretory pathway and the endosomal pathway. The secretory pathway starts with the expression of a protein. The newly expressed protein is then transported to the endoplasmic reticulum (ER), where it is processed and then brought to the Golgi for further modifications. The Golgi is built of several cisternae, maturing from the cis-Golgi via the medial-Golgi to the trans-Golgi and TGN.
From the TGN, the protein is either secreted at the PM, or delivered to organelles belonging to the endosomal pathway. The endosomal pathway starts with endocytosis at the PM. After fission, the endocytosed cargo is transported to an early endosome (EE), where it is either recycled back to the PM or delivered via a multi-vesicular-body (MVB), to late endosomes (LE) to and then lysosomes for digestion.
In the following I will elucidate the two membrane trafficking pathways in more detail (see also fig.
1.1-1). All mentioned data belong to studies on mammalian cells and yeast. Afterwards I will introduce Rab proteins and tethering factors. In the last part of the introduction I will focus on Dictyostelium, the model organism used for this study and highlight the similarities and differences to human and yeast.
Figure 1.1-1: Membrane trafficking in a mammalian cell. The endosomal pathway starts with endocytosis, an early endosome (EE) is formed. From the EE the cargo is either recycled back to the PM via recycling endosome and recycling vesicle or transported to multivesicular body (MVB), from where it gets to the late endosome (LE) and it final destination the lysosome. The post-lysosome (PL) is marked with a dashed line, because it exists only in Dictyostelium. The secretory pathway includes transport of cargo from the endoplasmatic reticulum (ER) to the Golgi and further to the trans Golgi network (TGN) for final secretion at the plasma membrane.
Arrows indicate all possible membrane trafficking pathways between the different organelles.
1.1.1 The secretory pathway
Newly synthesised proteins are transported from the ER to the cis-Golgi. These proteins bear a sorting signal in their cytosolic tail, which is recognised by cytosolic adaptors and coat proteins of the COP II complex. The COPII complex forms a coat out of two heterodimers, Sec23-Sec24 and Sec13-Sec31, which are recruited to ER exit sites by the small GTPase Sar1. Prior to arrival at the cis-Golgi the vesicle is uncoated for fusion. After vesicle fusion with the cis-Golgi, the protein matures through the medial and the trans Golgi stacks to the TGN. Additionally, a part of the vesicle content is recruited back to the ER. Retrograde Golgi-to-ER transport is mediated by COP I coated vesicles. The COP I- complex is recruited to membranes by ARF (ADP-ribosylation factor) GTPases. ARF proteins are also important for structure and function of the Golgi complex (Gillingham and Munro, 2007). They mediate as well intra-Golgi retrograde transport.
The TGN is a tubular network that originates from the trans-Golgi stack. At the TGN, the secretory pathway is connected to the endosomal pathway. Proteins are either delivered to the different types of endosomes (see the endosomal pathway) or they leave the TGN in a secretory vesicle for secretion at the PM. This can either be at a random place, or highly regulated as in polarised epithelial cells, where vesicles are directed to the apical or the basolateral surface.
1.1.2 The endosomal pathway
The endosomal pathway begins with the internalisation of PM proteins, lipids, extracellular material, particles, ligands and receptors by endocytosis. Respectively, endocytosis can be either be a way of feeding when engulfing bacteria or other nutrient rich substances and at the same time a way to regulate cell signalling by controlling the number of receptors respectively ligands at the PM (Grant and Donaldson, 2009; Sorkin and von Zastrow, 2009).
Regarding its numerous functions 5 different classes of endocytosis exist, (1) phagocytosis, the uptake of a particle > 0.5 µm, (2) macropinocytosis, (3) clathrin-mediated endocytosis, (4) caveolae-mediated endocytosis and (5) clathrin- and caveolae independent endocytosis. The classes two to five can be summarised as pinocytosis, which is generally known as the uptake of fluid (Conner and Schmid, 2003; Nichols, 2003). In the following I will not distinguish between the different types of endocytosis, because the maturation of the resulting endosome is similar.
A well-studied examples of clathrin-mediated endocytosis is the endocytosis of the transferrin receptor (TFR) and low-density lipoprotein (LDLR). Caveolae-mediated endocytosis used by glycosphingolipids and some viruses for cell entry (Mayor and Pagano, 2007; Sandvig et al., 2008). In contrast to clathrin-mediated endocytosis, Caveolae-mediated endocytosis forms flask-shaped invaginations of the PM. For both types of endocytosis vesicle scission depends on the GTPase
dynamin. Shortly afterwards vesicle coat complexes are shed and the vesicle it fuses with the EE. The EE is characterised by the presence of Rab5 and early endosome antigen 1 (EEA1). It is an important sorting station. Cargo can be delivered from the EE to a LE, destined for degradation, to the TGN or it can be recycled back to the PM. Proteins as sorting nexins are important regulators at this step. They prevent most of the cargo from degradation and sort it into the recycling pathway. Recycling happens via two different ways. For rapid recycling vesicles bud from the EE and move directly back to the PM. Proteins using the second recycling pathway, the slow recycling are first transported to the recycling endosome (RE) and then to the PM. The RE originates from early endosomal tubules. It is positioned juxtanuclear and extends tubules on microtubules into the cell periphery. Those tubules lose Rab5 and acquire Rab11, a RE localised Rab. From the tubules vesicles bud and fuse with the PM (Sonnichsen et al., 2000). The non-tubular part of the EE matures into multi-vesicular-bodies (MVB) (Maxfield and McGraw, 2004). MVBs contain for example many signal-receptors that are targeted for degradation by ligand-induced ubiquitylation. Sorting of those receptors is mediated by the endosomal-sorting complex required for transport (ESCRT)-complex (Nickerson et al., 2007; Raiborg and Stenmark, 2009).
From the MVB, cargo is delivered to the LE. LE are characterised by the presence of Rab7 and Rab9.
From the LE cargo can either be transported back to the TGN or to the lysosome for final degradation.
The endosomal pathway, ends in the lysosome. Lysosomes are fill with lysosomal enzymes for cargo digestion. The two main classes of lysosomal enzymes are hydrolases and integral lysosomal membrane proteins (LMPs). Lysosomal hydrolases are essential for bulk degradation, degradation of the extra cellular matrix, pro-protein processing, antigen processing and initiation of apoptosis (Dell'Angelica et al., 2000). LMPs are involved in acidification of the lysosomal lumen, transport of degradation products to the cytosol, protein import from the cytosol and membrane fusion (Eskelinen et al., 2003). Most of the LMPs reach the lysosome via the endosomal pathway. Secretion from the TGN to the PM is followed by re-endocytosis and subsequent arrival in the lysosome, where the enzymes are activated. Activation depends on the pH, which drops in the lysosome to pH 4 - 5.
1.2 Rab proteins, a member of the superfamily of small GTPases
Small GTPases are molecular switches that regulate a large number of cellular and developmental events. These are differentiation, cell division, nuclear assembly and membrane trafficking. For regulation of these processes they alternate between an active and an inactive form, which depends on their nucleotide status. Activation of a GTPase (e.g. a Rab) takes place by nucleotide exchange from GDP to GTP through a guanine nucleotide exchange factor (GEF). Rab-GTP is able bind its effector and to be recruited to membranes. Membrane binding takes places via hydrophobic geranylgerany groups that are attached to one or two carboxy-terminal Cys residues (Soldati et al., 1994; Ullrich et al., 1994; Ullrich et al., 1993). Subsequently, the protein is activated by hydrolysis of GTP, mediated by a GTPase-activating protein (GAP). Rab-GDP dissociates from its effector back into the cytosol.
Now, the effector triggers the next step in the pathway. The cytosolic Rab-GDP is recognised by a Rab GDP dissociation inhibitor (GDI) and. Interaction of Rab-GDP with a membrane-bound GDI displacement factor (GDF) starts a new cycle of GTP hydrolysis (Stenmark, 2009).
Figure 1.2-1: Activation-inactivation cycle of Rab proteins. In its inactive form, cytoplamic Rab-GDP is bound to its GDP dissociation inhibitor (GDI). To enable Rab to bind to its effector, a guanine nucleotide exchange factor (GEF) binds to Rab-GDP and catalyses the
exchange of GDP to GTP. Rab-GTP binds its effector.
Activation of Rab-GTP is catalysed by a GTPase-
activating protein (GAP) that hydrolyses to GTP to GDP.
Rab-GDP dissociates from its effector, which can now trigger a new step. Cytosolic Rab-GDP starts a new cycle.
Adapted from (Stenmark, 2009)
The superfamily of small GTPases includes Rab (Ras analog in brain), Ras, Rac / Rho, Arf and Ran proteins. Rab proteins are the main regulatiors of membrane trafficking. They act at all stages of the endosomal and secretory pathway. By interaction with their effector proteins they control a large number of processes as vesicle formation, recycling, transport, tethering and fusion. Up to now, more then 60 members of the human Rab family have been characterised and localised to discrete intracellular membranes (Stenmark, 2009). In the following, the most important functions will be highlighted.
In most cases, several Rabs localise to one donor organelle to ensure the transport to different acceptor organelles. Rab4, Rab5 and Rab35 localise to EE, whereas Rab5-GDI is already essential for clathrin- mediated endocytosis of TfR (McLauchlan et al., 1998). Each Rab protein is found in a distinct patch.
Rab4 and Rab35 regulate the rapid recycling from the EE to the PM. Rab11 and Rab22a control the slow recycling from the EE via the RE to the PM. Interfering with Rab11 or its regulators inhibits recycling and often alters the position of the RE. Rab22a is implicated into cargo transport from the EE to the RE. Rab22a depletion blocks TfR recycling (Sonnichsen et al., 2000). Several Rab proteins localise to the TGN to regulate vesicle formation and sorting. Rab8 and Rab10 control for example the apical versus basolateral trafficking. Rab8 organises the AP1B-dependent delivery of basolateral vesicles, whereas overexpression of Rab8 blocks basolateral but not apical transport (Ang et al., 2003;
Kroschewski et al., 1999; Musch et al., 2001). Overexpression of Golgi-localised Rab10 results in inhibition of TGN-to-PM transport and mis-sorting of basolateral cargo to the apical membrane (Schuck et al., 2007).
An example for Rab-dependent vesicle budding is Rab9 on LE. Rab9 positive membrane patches on the LE show a high concentration of mannose 6 phosphate receptor (M6PR). This is induced by the interaction of Rab9 with its effector Tip47, a sorting adaptor with increased affinity for the M6PR in its Rab-bound form. The vesicle buds off and transports the M6PR back to the TGN for another round of lysosomal sorting (Carroll et al., 2001; Saftig and Klumperman, 2009).
In many cases, vesicle transport depends on the interaction of a Rab protein with a molecular motor.
Rab6 for example, interacts with its effector rabkinesin6 on TGN-derived vesicles. So it influences the rate of exocytosis by enhancing kinesin-dependent transport of secretory vesicles (Grigoriev et al., 2007). Also, Rab27a is involved in vesicle transport. It links melanosomes to myosin Va via the adaptor protein melanophilin (Wu et al., 2002). Furthermore, Rab27a is important for SNARE-
mediated docking of exocytic dense-core vesicles to the PM. In that case, Rab27a binds via its effector granuphilin to Sec1-related munc 18-1 (syntaxin binding protein-1). Rab3a is implicated in the same process (Fukuda, 2008). The role of Rab proteins in vesicle tethering will be discussed in detail under 1.3 "Tethering complexes".
1.3 Tethering complexes
Tethering is defined as the first contact between two membranes before SNARE-mediated fusion.
Activated Rabs (and perhaps other GTPases) regulate the recruitment of tethering complexes to the membrane and participate in formation of a tether-binding site followed by SNARE interaction and fusion of the membranes (Grosshans et al., 2006; Peplowska et al., 2007). It has been proposed that SNAREs determine the precise place of the fusion. This is in contradiction with the homogeneous distribution of SNAREs on some membranes even though Lang and colleagues propose that SNAREs assemble in membrane rafts. They showed that syntaxin1 and 4 (t-SNAREs) clusters define docking and fusion sites for secretory vesicles, but could not co-localise these clusters with a raft maker Thy-1 (Lang et al., 2001).
In parallel it has been proposed that tethering complexes specify the place of fusion. The tether bridges between vesicle and target membrane, which impose membrane selectivity. Subsequently SNAREs come close enough to engage the opposing membranes for fusion (Lupashin and Sztul, 2005).
Up to date, several tethering factors have been described. They fall into two broad categories: 1. long putative coiled-coil proteins and 2. multi-subunit complexes. Members of the first group are Uso1p/p115, GM130, giantin, golgin84, golgin97 and EEA1. Members of the second group are the exocyst, COG (conserved oligomeric golgi), GARP (golgi-associated retrograde protein), HOPS (homotypic fusion and vacuole protein sorting), CORVET (class C core vacuole / endosome tethering), TRAPPI (transport protein particle), TRAPPII and Dsl1. The exocyst, COG and GARP belong to the quatrefoil complexes, because the number of complex subunits is always a multiple of four. Subunits of these complexes are predicted to have a common N-terminal domain. Figure 1.3-1 depicts the N-terminal domain with always two short stretches of potential coiled-coil or amphipatic helix. This suggests that these complexes have a similar mode of action or of assembly (Whyte and Munro, 2002).
In the remaining part of this chapter I will concentrate on the role of the multi-subunit complexes and give a brief description of each. The role of the exocyst will be discussed separately and in more depth in 1.3.7 "The exocyst complex". Figure 1.3-2 summarises the localisation of the different complexes.
All protein names used are taken from yeast, because tethering factors have been studied best in this organism. For better understanding, the mammalian name is given in brackets.
Figure 1.3-1: Subunits of the human COG complex and human exocyst blotted for the probability to form coiled coils. x-axis: protein length, y-axis: probability of a coiled-coil being at each residue of the protein, as determined by the algorithm of Lupas (Lupas, 1996). For details see: (Whyte and Munro, 2002)
Figure 1.3-2: Tethering complexes act during various steps of the yeast secretory pathway.
Adapted from (Whyte and Munro, 2002)
1.3.1 The COG complex
The COG complex is an octameric complex. In yeast and mammalian cells, the subunits are named Cog1p to Cog8p. They group into two lobes, Cog1p-Cog4p and Cog5p-Cog8p. In yeast, Cog1p is supposed to bridge between the two lobes (Fotso et al., 2005). Recently, the main part of the Cog2p structure was solved (Cavanaugh et al., 2007). It is composed of six-helix bundle with conserved surface features. This resembles the crystal structure of the exocyst subunits.
The COG complex acts as a tether at the Golgi apparatus. Vesicles that recycle within the Golgi or vesicles delivered from later, endosomal compartments are tethered by the COG complex to the cis Golgi (Whyte and Munro, 2002). COG interacts with Ypt1p (Rab1), intra-Golgi SNAREs and the COPI vesicle coat (Lupashin and Sztul, 2005). Mutations of a COG subunit cause a congenital disorder of glycosylation (CDG) in humans (Freeze, 2007).
1.3.2 The GARP complex
The GARP complex contains four subunits, Vps51p, Vps52p, Vps53p and Vps54p (vacuolar protein sorting). Recently, a mammalian GARP complex has been identified. In yeast, GARP appears to have a dual function in tethering of vesicles derived from EE or from prevacuolar / lysosomal compartments to the Golgi and the TGN. The GARP complex is an effector of Ypt6p (Rab6), which localises to the trans-Golgi. It is also known that the subunit Vps51p binds to the N-terminus of the late Golgi / TGN SNARE Tgl1. (Cai et al., 2007; Sztul and Lupashin, 2006). In yeast, mutation of the complex results in the missorting of 70% of the vacuolar carboxypeptidase Y, as well as in the missorting of late Golgi membrane proteins to the vacuole (Lupashin and Sztul, 2005).
1.3.3 The HOPS complex
The HOPS complex also known as Class C Vps complex was identified through characterisation of the numerous yeast mutants that show defects in sorting of proteins to the vacuole. It is involved in Golgi-to-endosome transport, homotypic fusion of vacuoles and fusion of vesicles to the vacuole.
Recently, human homologues of the yeast subunits have been characterised and found to be localised in a complex on late endosomes / lysosomes.
The complex consists of six subunits called Vps11, Vps16, Vps18, Vps33, Vps39 and Vps41. Vps39 acts as an effector of Ypt7p (Rab7) and functions as a GEF. It binds Ypt7p-GDP and converts it to Ypt7-GTP to promote vesicle tethering. The subunit Vps33 is a Sec1 homolog. It binds the vacuolar SNARE Vam3p (Whyte and Munro, 2002). Moreover, phosphorylation of the subunit Vps41 is important for the organisation of vacuole fusion sites. Non-phosporylated Vps41 co-localises with other HOPS subunits around the yeast vacuole in punctuate structures that correspond to in vivo fusion sites. A phosphomimetic mutant of Vps41 induces a fusion defect. Both phenotypes can be overcome by Ypt7p overexpression, indicating that the localisation of Vps41 is determined by Ypt7p (Cabrera et al., 2009).
1.3.4 The CORVET complex
The CORVET complex, the most recently identified an endosomal-tethering complex shows a high homology to the HOPS. The two complexes share four subunits, Vps11, Vps16, Vps18 and Vps33.
The subunits are able to interconvert by dynamic subunit exchange. Vps3 and Vps8 are the CORVET specific subunits, whereas Vps3 has a high functional homology to Vps33. It binds Vps21-GDP (a homolog of the endosomal Rab5) and promotes the nucleotide exchange to Vps21-GTP (Cai et al., 2007; Peplowska et al., 2007).
1.3.5 The TRAPPI and II complex
The TRAPP complexes are large protein complexes functioning in later stages of ER-to-Golgi traffic.
TRAPPI is composed of seven subunits Bet3p, Bet5p, Trs20p, Trs23p, Trs33p, Trs31p and Trs85p with a total size of about 300 kDa. It is required for ER-to-Golgi transport and binds to COPII vesicles. TRAPPII contains three additional subunits Trs65p, Trs120p and Trs130p, which results in a size of about 1000 kDa. TRAPPII acts between late Golgi and endosomes. At least seven of the TRAPP subunits are well conserved in mammals and present in a large complex.
Like the HOPS complex, both TRAPP complexes have a GEF activity. TRAPPI acts on Ypt1p (Rab1). TRAPPII promotes the nucleotide exchange of Ypt31p / 32p (Rab11). The two TRAPPII complex specific subunits Trs120p and Trs130p are essential for this process and inhibit at the same time the Ypt1p GEF activity of TRAPPII (Markgraf et al., 2007; Morozova et al., 2006; Sacher et al., 2008)
1.3.6 The Dsl1 complex
The Dsl1 complex is a trimeric complex witht the subunits Dsl1p, Tip20p and Sec39p. All three subunits are ER-localised peripheral membrane protein essential for retrograde ER-Golgi trafficking.
The complex is known to interact with several ER-localised SNAREs Use1p, Ufe1p and Sec20p (Kraynack et al., 2005). Recently, the group of Hughson proposed that the Dsl1 complex, the exocyst and the COG complex have a common ancestor. Based on structural analyses they showed that Tip20p and the exocyst subunit Exo70p share a core structure of helix-bundle domains (Tripathi et al., 2009).
1.3.7 The exocyst complex
In 1980, Peter Novick and others published a genetic screen of proteins that function in the secretory pathway in yeast (Novick et al., 1980). They called them the "sec"-proteins. 15 years later TerBush and colleagues showed that six of those proteins, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15 plus two additional ones, Exo70 and Exo84 assemble into an octameric complex, the exocyst complex. This complex was called the exocyst complex, because in yeast it tethers post-Golgi secretory vesicles to the PM (TerBush and Novick, 1995). Interference with a subunit leads to a block in secretion and growth arrest. TerBush and colleagues showed that the exocyst is important for polarised delivery of vesicles to sites of bud growth. It provides additional membrane material for bud growth In the beginning of cytokinesis, the complex localises at the bud tip, afterwards it is distributes homogenously at the PM of the growing bud. At late stages of cytokinesis, just before abscission it is found at the mother-bud-neck (Finger et al., 1998).
Within the last 15 years the exocyst has been an object of intense studies. The complex is evolutionary conserved from yeast to human. Homologs of the exocyst subunits have been found in various organisms. Also in plants, subunits of the exocyst have been identified. In contrast to yeast, most of
the putative plant exocyst subunits exist in multiple copies. Lately, Hala and colleagues demonstrated that several subunits function in a complex. They are involved in polarised cell growth, like root hair elongation, hypocotyl elongation and pollen tube growth (Eckardt, 2008; Elias et al., 2003; Hala et al., 2008; Synek et al., 2006).
Furthermore, the importance of the complex has been underlined by fact that knock-out of Sec8 is early embryonic lethal in mice (Friedrich et al., 1997).
In the following, I will describe the different features of the complex, like its structure and how is it targeted to the PM and assembed. Furthermore, I will show examples for the function of the complex in processes such as vesicle transport in polarised epithelial cells, secretion of Glut4 vesicles in adipocytes, focal exocytosis during cytokinesis and neurite outgrowth.
1.3.7.1 Structure of the exocyst complex
The exocyst subunits are large (70 -150 kDa) hydrophobic proteins with a low solubility. These conditions made the structure determination especially difficult. But in recent years, the crystallisation of full-length yeast Exo70, the C-terminus of yeast Exo84, the C-terminus of yeast Sec6 form yeast as well as the C-terminus of Drosophila melanogaster Sec15 could be achieved. All four proteins form highly elongated structures. Exo70 forms a 160 Å-long rod composed of α-helices. The C-terminus of Exo84 C forms an 80 Å-long rod, which has surprisingly the same fold as Exo70 N-term even though the sequence similarity is below 10%. The crystal structures of Sec6 C-term and Sec15 C-term fold revealed α-helical rods, which resemble the Exo70 structure (see Figure 1.3-3) (Dong et al., 2005;
Hamburger et al., 2006; Sivaram et al., 2005; Wu et al., 2005).
Based on this observation, Dong and colleagues suggested that the exocyst subunits assemble against each other in an end-on manner resulting in an elongated rod-like structure. The rod-like structure of the exocyst is placed between the two membranes to make a first contact before subsequent interaction of SNAREs and membrane fusion. This hypothesis was supported by quick-freeze/deep-etch EM of the exocyst complex. Figure 1.3-4 shows the EM images and the a model by Munson and Novick (Munson and Novick, 2006).
Figure 1.3-3: Crystal structure of Exo70, Exo84 C-terminus, Sec15 C-terminus, and Sec6 C- terminus. The four proteins exhibit a common backbone fold. (Munson and Novick, 2006)
Figure 1.3-4: The exocyst assembles to a rod like structure. (a and b) Deep etch EM pictures of the exocyst. (c) Model of exocyst assembly and tethering of vesicle to PM. (Munson and Novick, 2006)
1.3.7.2 Assembly and targeting of the exocyst complex
Assembly and targeting of the exocyst complex is mainly regulated by a number of GTPases. The assembly of the complex has been studied in most detail in mammalian cells. The complex forms two sub-complexes. One contains the subunits Sec5, the other one Exo84. Sec5 and Exo84 interact with RalA, a small GTPase that does not exist in yeast (Moskalenko et al., 2003). Through this interaction RalA regulates the full assembly of the complex. In Ral loss-of-function mutants subunits belonging to the two sub-complexes, as Sec6, which binds to Sec5 and Sec10, which binds to Exo84 are not able anymore to interact.
The targeting process of the yeast and human exocyst follows the same principal, but involves different GTPases. Therefore I will describe the targeting of the yeast exocyst complex in detail and highlight the differences to mammals afterwards.
1.3.7.3 Targeting of the exocyst complex to sites of exocytosis
It was proposed that the majority of the exocyst subunits are targeted to the PM in a pre-assembled state. Therefore, they the sub-complex binds to the cargo vesicle and is transported with the vesicle to the PM. This depends on the peripheral exocyst subunit Sec15. Sec15 binds Sec2, the GEF of Sec4 and Sec4 at the same time on the vesicle. This dual interaction brings Sec4 and Sec2 close enough together for nucleotide exchange of Sec4 (Medkova et al., 2006; Zajac et al., 2005). Sec4-GTP binds as well Myo2, a myosin V, which transports the vesicle to the PM along polarised actin cables. At the PM, Sec15 and Sec4 form a patch at the site of secretion. Overexpression of Sec15 inhibits cell growth and leads to the accumulation of secretory vesicles in the bud (Guo et al., 1999b).
The assembly of the complex depends on interaction between the single subunits. Several pair-wise interactions were shown between subunits by Yeast 2-Hybrid screens and immunoprecipitations.
Assembly of Exo84 into the complex requires Sec5p and Sec10p. Exo84 is mislocalised in sec5
mutants (Guo et al., 1999a). Mutation of Exo84 lead to defects in invertase secretion and accumulation of secretory vesicles (Guo et al., 1999a). In general, each subunit apart from Sec3 is essential in yeast.
Figure 1.3-5: Thin EM section of yeast. (A) wild type yeast. (B) Exo84 depleted yeast. Accumulation of secretory vesicles in the cell. (Guo et al., 1999a)
On arrival at the PM, the exocyst complex is targeted for vesicle tethering to the place of secretion.
There has been a long series of investigations about the subunits responsible for targeting of the complex. In the late 90ties the group of Peter Novick proposed Sec3 to be the "landmark" at the PM.
They showed that Sec3 localises at the PM independently of the actin cytoskeleton and a of functional secretory pathway to the PM. Other subunits like Sec8 were mislocalised in sec3 mutants (Finger et al., 1998), proposing that there localisation depends on Sec3. Two small GTPases have been proposed to localise Sec3 independently of actin to the PM, Cdc42 and Rho1. Sec3 and Cdc42-GTP interact directly and co-localises. A cdc42 mutant shows a randomised exocytic pattern. Cdc24, the Cdc42 GEF seems to regulate targeting of Sec3 to exocytic sites seems (Zhang et al., 2001). Sec3 interacts a well with the Rho1. Rho1 mutants change Sec3 localisation. Loss of Rho1-Sec3 interaction abolishes correct localisation of all the other exocyst subunits except of Exo70 (Guo et al., 2001). Exo70. The localisation of Exo70 does not depend on Sec3. Exo70 interacts with another small GTPase, Rho3.
Rho3 and Exo70 co-localise at the PM. Rho3 mutants cells are large and rounded with an aberrant cytoskeleton.
Boyd and colleagues made FRAP experiments measuring the recovery rates of the subunits at the PM to define, which of the two subunits is the "true" landmark. They found two types of recovery. Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 showed a fast recovery close to the one of Sec4. In contrast, Sec3 and Exo70 showed a slower recovery rate. This led them to conclude that the first set of proteins is transported on the vesicle along actin cables to the PM and the second set of proteins is recruited directly to the PM. Exo70 seems to use both pathways, indicating that both proteins could function as a landmark (Boyd et al., 2004). The group of Brennwald further investigated the role of Sec3 and Exo70 at the PM. The Exo70-binding deficient mutant rho3-V51 has a pronounced secretion phenotype, even though the other exocyst subunits are still polarised normally (Roumanie et al., 2005). In addition they showed that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 targeting pathways. A rho3-V51, sec3ΔN double mutant showed the same secretion phenotype as rho3-V51. Cdc42, Sec4, Myo2 and the actin cytoskeleton are still polarised, indicating that there might be a further way to target the exocyst subunits to the PM. In following, the group of Guo discovered that C-terminus of Exo70 and the N-terminus of Sec3 directly interact with PI(4,5)P2 in the PM (He et al., 2007; Zhang et al., 2008). Disruption of the Exo70 - PI(4,5)P2 leads to Exo70
mislocalises and loss of membrane association. Even though the remaining subunits are still localised.
The same holds true for Sec3. However, in the double mutant of exo70-45, sec3ΔN the exocyst subunits were completely depolarised and unable to anchor at the PM. These suggests that the interaction of Sec3 and Exo70 with PI(4,5)P2 is the key issue in exocyst stabilisation at the PM and that both proteins function in concert. Figure 1.3-6 summarises all known interactions of the exocyst directly with the PM.
Figure 1.3-6: Exocyst-mediated vesicle tethering to the PM is regulated by small GTPases (red circles) and interaction of the exocyst subunits Sec3 and Exo70 with PIP2 at the PM. (He and Guo, 2009)
The double mutant, sec3ΔN, exo70ΔC is unviable, even though ExoΔC is still able to bind to PI(4,5)P2, but not to Rho3 (Hutagalung et al., 2009). The actin cytoskeleton remains polarised and transport of Sec4 to the PM is intact, but Sec5 is complete mislocalised. Therefore, Hutagalung and colleagues conclude that, besides the interaction of Exo70 with Rho GTPases and PI(4,5)P2, it seems also to be important for the assembly of the complex at the PM.
In parallel to the function of Sec3 and Exo70, the yeast cell developed further mechanisms to correct ensure tethering and fusion by acting directly on the SNARE complex. The SNARE complex in yeast is formed by the t-SNARE Sso1p/2p and Sec9p together with the v-SNARE Snc1p/2p (Brennwald et al., 1994; Grote et al., 2000). Sec1, a SNARE-binding protein that stimulates SNARE-mediated membrane fusion in vitro co-immunoprecipitates with the exocyst and (Carr et al., 1999; Scott et al., 2004). Overexpression of SEC1 or SEC4 rescues deletions of the essential exocyst subunits Sec5 and Exo70 and the growth defect of sec3Δ cells, indicating that defects in exocyst assembly can be overcome by stimulation of SNARE formation (Wiederkehr et al., 2004). Expression of multi-copy SSO2 or SEC9 plasmids improve the sec3Δ growth defect, confirming this hypothesis.
Another group of proteins, the lethal giant larvae (Lgl) family proteins Sro7 and Sro77, have a similar effect. Lgl was first identified in Drosophila as a tumor suppressor. Sro7 and Sro77p interact with
Exo84 and Sec9p. Overexpression of SRO7, SSO1/SSO2, SEC9 and SEC1 rescue an exo84 mutant.
In mammals, targeting of the exocyst complex to exocytic sites functions in principal similarly. In both organisms Exo70 interacts with PI(4,5)P2 at the PM (Liu et al., 2007). But in contrast to yeast, mammalian Exo70 interacts with the small GTPase TC10 and not with Rho3 (Inoue et al., 2003).
Also, mammalian Sec3 has no Rho1-binding domain (Matern et al., 2001). Mammalian Sec15 binds to Rab11 instead of the Sec4 homolog Rab8. However, Sro7/77-mediated exocyst-SNARE complex interaction is found as well in mammalian cells. The mammalain homolog of Sro7/77 is Lgl1/2. A comparison of both organisms is shown in figure 1.3-7.
Figure 1.3-7: Comparison of exocyst-mediated vesicle tethering to the PM in yeast and mammals. (Brennwald and Rossi, 2007)
1.3.7.4 The exocyst in focal exocytosis
The exocyst is involved in various events of focal exocytosis including neurite outgrowth, cytokinesis, transport of Glut4-positive vesicles to the PM and vesicular trafficking in polarised epithelial cells.
1.3.7.4.1 Exocyst-mediated vesicle trafficking in polarised epithelial cells
In polarised epithelia cells, the exocyst is important for the regulation of membrane trafficking to the apical and basolateral pole. Subunits of the exocyst localises to the TGN, to RE and to tight junctions.
Sec10 for example is found at RE. It interacts with the small GTPase Arf6 (Prigent et al., 2003).
Overexpression of Arf6 results in a re-localisation of Sec10 from RE to the PM. At the PM, Arf6 triggers membrane ruffle and filopodia formation. Prigent and colleagues propose that the exocyst is needed for Arf6-induced membrane recycling during cell spreading.
Another exocyst-related protein found on RE is Rab8, the mammalian homolog of Sec4. Rab8 co- localises with the clathrin adaptor AP-1B on TGN to RE vesicles. No direct interaction between the
exocyst, specially Sec15 has been demonstrated, but overexpression of µ1B in AP-1B-negative cells enhances the recruitment of Sec8 and Exo70 to the perinuclear TGN-RE region (Folsch et al., 2003).
This is of special interest for this thesis, because we will report that in Dictyostelium, Rab8a interacts with Sec15.
The exocyst subunit Sec15 localises as well to Rab11-GTP-positive RE, but overexpression of Sec15 has no influence on RE-mediated recycling of the transferrin receptor (TfnR) (Zhang et al., 2004). In addition, Sec15 is involved in basolateral-to-apical transcytosis, because down-regulation of Sec15 impairs this trafficking pathway (Oztan et al., 2007).
Already in 1998 Grindstaff and colleagues suggested that the exocyst plays a role in transport to the basolateral membrane (Grindstaff et al., 1998). Addition of function-blocking Sec8 antibodies resulted in delivery of basolateral proteins to the apical membrane, but delivery to the apical membrane was not affected.
Later, it was shown that the exocyst is especially enriched at tight junctions, an area of active exocytosis. Sec8 co-fractionates with proteins of the tight junction complex, Zonula-1 (ZO-1) and Zonula-2 (ZO-2),. Two other components of tight junction complex, E-cadherin and nectin-2α recruit the exocyst to the PM (Yeaman et al., 2004). In Drosophila, was shown that DE-cadherin trafficking from RE to the adherens junctions is blocked in Sec5 and Sec6 mutants. Instead, DE-cadherin accumulates in large Rab11-positive endosomes (Langevin et al., 2005). Correct localisation of adherence junction proteins is mediated by Exo84. Absence of Exo84 leads to mislocalisation of Crumbs an apical trans membrane proteins and the localisation of Rab11-positive RE is disrupted (Blankenship et al., 2007).
1.3.7.4.2 Exocyst-mediated transport of Glut4 vesicles to the PM
Glucose stimulates insulin secretion from large dense core vesicles in pancreatic β cells. This leads at the same time to insulin-mediated recruitment of the glucose transporter 4 (Glut4) from recycling vesicles to the PM, which results in glucose uptake.
Targeting and fusion of Glut4 vesicles to the PM is mediated by the exocyst. After insulin secretion the small GTPase TC10 recruits Exo70 to the PM (Inoue et al., 2003). Overexpression of Exo70 results in 48% increase of glucose uptake. In contrast, overexpression of truncated Exo70 N-term that is unable to bind TC10 shows a 40% reduction of glucose uptake. Sec6 and Sec8 are also implicated in Glut4 vesicle transport and fusion. Sec6 localises to insulin-positive vesicles and Sec8 is found at the PM. Knock-down of both subunits results in reduced insulin-stimulated glucose uptake (Tsuboi et al., 2005). Tethering and recruitment of the vesicles seems to be regulated by the small GTPase RalA (Chen et al., 2007). RalA-GTP is required for glucose uptake. Additionally, it binds to Myo1c.
Deletion of the Myo1c motor domain results in less RalA at the PM, indicating that prior to tethering Myo1c transports the Glut4 vesicle including part of the exocyst complex and RalA along actin cables to the PM.
1.3.7.4.3 Exocyst-mediated vesicle delivery during cytokinesis
Cytokinesis is the process of division and separation of two cells during meiosis or mitosis.
Cytokinesis consists of two steps. At first, an acto-myosin rings forms close to the equatorial cortex of
the dividing cells, then the ring constricts to form the cytokinetic furrow. At the end of constriction, the two daughter cells are still attached by a thin cytoplasmic bridge. Final abscission separates the cells completely (Glotzer, 2001; Schweitzer and D'Souza-Schorey, 2004).
Ring constriction and formation of the cytokinetic furrow depend on exocyst-mediated membrane delivery. Mutations in exocyst subunits lead as well to defects in cytokinesis (Cascone et al., 2008;
Gromley et al., 2005). In more detail, Cascone and colleagues showed that the exocyst, regulated by RalA and RalB is important for two steps in cytokinesis. Firstly, RalA controls assembly and tethering of the complex at the cytokinetic furrow. Whereas Sec6 localises to the vesicle and Sec5 to the PM.
Secondly, RalB recruits the exocyst to the midbody to control abscission.
Another exocyst-related small GTPases implicated in cytokinesis is Rab11. Rab11-positive vesicles enrich close to the cytokinetic furrow in ana-telophase cells. Rab11 recruits several effectors to the vesicle, like FIP3 (family of Rab11-interacting proteins) and part of the exocyst. FIP3 accumulates with Rab11 in the cytokinetic furrow and seems to be important for targeting of the vesicles to the midbody. Depletion of FIP3 induces loss of the perinuclear recycling endosome compartment (Inoue et al., 2008). Whereas knock-down of Rab11 results in defects in furrow regression and cell scission (Skop et al., 2001).
1.3.7.4.4 Exocyst-mediated neurite outgrowth and synaptogenesis
The exocyst is required for neurite outgrowth and synaptogenesis, but not for secretion of synaptic vesicles. The exocyst is highly expressed in terminals of neurite outgrowth and marks sites of synaptogenesis along axons (Hazuka et al., 1999). Drosophila loss-of-sec5 mutant do not form neuromuscular junctions, because of a defect in vesicle trafficking (Murthy et al., 2003). The vesicles are not tethered to the PM and fail to fuse. A loss-of-sec15 Drosophila mutant is able to perform neurit outgrowth but forms synapses with the wrong partners (Mehta et al., 2005). Metha and colleagues propose that Sec15 is important for the selective delivery of cell adhesion and signalling molecules, which define synaptic specificity, indicating that each exocyst subunit has a specific function.
1.3.7.4.5 Exocyst-mediated invadopodia formation
The role of the exocyst in invadopodia formation is of special interest as it has been linked to metastasis and cancer cells invasion. Invadopodia are specialised filopodia, which are able to form membrane protursions with matrix proteolytic activity. Invasive cancer cells form invadopodia to penetrate the extra cellular matrix (ECM) or invade other tissues by matrix degradation through metalloproteases (MMP). The formation of the invadopodia and the targeting of the MMP's to the tip of the filopodia is exocyst-dependent.
1.3.7.5 The exocyst in cell motility
In motile cells, clusters of exocytic events can be seen in the front of the cell close to the leading edge.
Targeting and assembly of the exocyst subunits to the leading edge is regulated by RalB (Letinic et al., 2009; Rosse et al., 2006). Disruption or knockdown of exocyst subunits results in less polarised cells and slower movement, showing that vesicle tethering to the leading edge is essential for directed, fast
cell motility. Furthermore, it has been shown that the exocyst is implicated in organisation of the actin network at the leading edge by actin on the Arp2/3 complex. The Arp2/3 complex nucleates new actin branches on existing actin filaments. In migrating cells, Exo70 is recruited to the leading edge, where it is essential for targeting of Arp2/3 to the PM by interaction of Exo70 with the Arp2/3 complex subunit Arpc1 (Zuo et al., 2006). Down regulation of Exo70 influences cell motility and blocks the formation of actin-based membrane protrusions.
1.4 The model organism Dictyostelium discoideum
Dictyostelium discoideum is a soil-dwelling amoeba belonging to the phylum Amoebozoa, subgroup Mycetozoa (Fiore-Donno et al., 2009). Dictyostelium has been isolated the first time in 1869 by Oskar Brefeld. 66 years later in 1935 K.B. Raper published the first article on the organism (Raper, K.B., 1935).
Under normal conditions the amoeba feeds on bacteria. Under starving conditions, or other types of stress it starts a unique asexual life cycle, also called differentiation. A stress factor triggers the release of the chemoattractant cAMP. This results in an cAMP gradient and aggregation of up to 100,000 cells by chemotaxis. The cells form a mound that finally develops into a fruiting body, with a stalk supporting a spore mass. Under more favourable conditions these spores geminate and re-start a new life cycle as vegetative cells dividing by mitosis. The whole development cycle lasts about 24 h.
Selection of mutants from the Dictyostelium wild isolate NC4 lead to the today commonly used lab strains AX2 and AX3. These are able to grow in axenic media (nutrient media without bacteria) due to their higher macropinocytic activity. AX2 and AX3 cells have a doubling time of about 10 h at 22°C.
The complete Dictyostelium genome has been published in 2005 (Eichinger et al., 2005). It has total size of about 34 Mb on six chromosomes with 4 - 7 Mb chromosome Additionally, there are multicopy extrachromosomal element of 90 kb encoding for the rRNA and the mitochondrial genome of 55 kb, present in about 200 copies. This totals about 12,500 protein-coding genes.
The sequenced genome revealed that a larger number of orthologues of human disease-related genes were found in Dictyostelium than in yeast. Dictyostelium being moreover haploid and easily genetically tractable lead to the fact that it is nowadays a widely used model organism in basic biomedical research. The main topics of research interest are membrane trafficking, cytokinesis, phagocytosis, chemotaxis, signal transduction, aspects of development such as cell sorting, pattern formation and cell-type determination (Urushihara, 2009). Further information, as well as the whole genome sequence are publically available on the website www.dictybase.org.
1.4.1 Membrane trafficking in Dictyostelium
Membrane trafficking in Dictyostelium is very similar to mammalian cells (Duhon and Cardelli, 2002;
Neuhaus et al., 2002). Additionally to the secretory and the endosomal pathway, Dictyostelium has a third major organelle involved in membrane trafficking, the contractile vacuole (CV). The function of the CV will be discussed in detail under 1.4.1.1 "The contractile vacuole". In this chapter, I will concentrate on the endosomal pathway.
Figure 1.4-1: Endosomal pathway of Dictyostelium. After endocytosis an early endosome with neutral pH is formed. By acidification and delivery of lysosomal enzymes the early endosome matures into a lysosome.
Digestion occurs. After about 30 min lysosomes start to fuse, re-neutralise and mature into post-lysosomes. The content of the post-lyosomes is exocytosed after about 60 min. During the course of maturation recycling takes place, here indicated by red arrows.
Figure 1.4-1 illustrates the endosomal pathway in a simplified way. After endocytosis an EE is formed. It is rapidly acidifies and matures into a lysosome. In contrast to mammalian cells, the Dictyostelium lysosome re-neutralises and forms a post lysosome (PL) that undergoes exocytosis.
Below, I will describe each step in more details, specially paying attention to the similarities and differences to the mammalian system.
Even though Dictyostelium endocytosis has mainly nutritive functions, clathrin-dependent and independent endocytosis also exist but are less well understood (Duhon and Cardelli, 2002; Neuhaus et al., 2002; O'Halloran and Anderson, 1992). The amoeba feeds either on phagocytosis or macropinocytosis. Proposed receptors for uptake are Sibs, SadA and Phg1 (Cornillon et al., 2008;
Cornillon et al., 2000; Fey et al., 2002). Phagosomes and macropinosomes mature in a comparable way, therefore we will discuss them together (Buczynski et al., 1997; Rupper and Cardelli, 2001).
During its first minute the EE is neutral and surrounded by a cytoskeletal coat. After dissociation of the coat it gets rapidly acidified via fusion of vesicles delivering the vacuolar ATPase (Maniak, 1999;
Maniak, 2001). Bogdanovic and colleagues identified the SNAREs syntaxin7, 8, Vti1 and Vamp7 that form a complex mediatng vesicle-endosome fusion (Bogdanovic et al., 2002). In parallel, membrane recycling back to the PM takes place. The group of P. Cosson localised a protein of 25 kDa, called p25, to RE (Ravanel et al., 2001). The slow recycling is mediated by myosinIB (Neuhaus and Soldati, 2000).
The acidic EE matures into a LE and then into a lysosome by acquiring lysosomal enzymes as cathepsin D and α-mannosidase. Digestion takes place and about 30 min later the lysosome matures into PL, therfore the proton ATPase and lysosomal enzymes are retrieved from the organelle and recycled back to earlier compartments. As a consequence the pH goes back to neutral. At the same time, Lysosomes undergo homotypic fusion and acquire proteins typical for the PL. These are vacuolin, a flottilin homolog and p80, a putative copper transporter (Jenne et al., 1998; Ravanel et al.,
