1 CHAPTER Introduction 1.1 Overview of the endocytic and secretory pathway of the eukaryotic cell One major difference between the eukaryotic and the prokaryotic cells is the presence of an intricate endomembrane system in the eukaryotic cells. This elaborate endomembrane system is responsible for the exchange of macromolecules between the eukaryotic cell and its environment. In this endomembrane system, the secretory pathway transports newly synthesized proteins, carbohydrates and lipids to the exterior of the cell, while the endocytic pathway is involved in the uptake of macromolecules into the cell. Transport along the secretory and endocytic pathway is a multistep process. It involves the formation of transport carriers containing distinct sets of cargo, the movement of cargo-loaded transport carriers between compartments, and the specific fusion of these transport carriers with the target membrane. The regulation of these processes requires the involvement of a complex collection of protein and lipid interactions. A general scheme of the endocytic and secretory pathway is shown in Figure (Bonifacino and Lippincott-Schwartz, 2003; Lee et al., 2004; Derby & Gleeson, 2007). 1.2 The endocytic pathway Endocytosis is the process by which cells internalize material from the outside the cell through the plasma membrane. Many important functions of a cell are mediated by endocytic mechanisms, including the uptake of nutrients, antigen presentation, maintenance of cell polarity and the regulation of cell-surface receptor expression. Viruses, toxins, and different micro-organisms also utilize the endocytic pathway to Plasma Membrane Clathrin non-clathrin mediated Lysosome Secretory Granule Late Endosome/ Multi‐Vesicular Body Early Endosome Recycling Endosome Immature Secretory Granule Trans‐Golgi Network trans Golgi Complex medial COPI cis ERGIC COPII ER Exit Site ER © OYS’09 Figure 1.1. Organization of the intracellular transport pathways. The scheme depicts the compartments of the secretory, lysosomal/vacuolar and endocytic pathways. Retrograde transport steps are indicated by pink arrows and anterograde transport steps are indicated by black arrows. Clathrin coats are in green, COPI in blue and COPII in red. The major organelles consists of the endoplasmic recticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus, early y, recycling y g endosome,, lysosome y endosome,, the late endosome/multi-vesicular body, and the plasma membrane. Adapted from Bonifacino and Glick, 2004. gain access to the cell. Endocytosis can be broadly divided into pinocytosis, which is the uptake of fluid and phagocytosis, which is the uptake of particles. Phagocytosis occurs mainly in specialized phagocytic cells such as macrophages, while pinocytosis is utilized by practically all eukaryotic cells for the uptake of extracellular fluid and solutes from the external environment. There are many ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. At least five different pathways of endocytic internalization are currently known. Phagocytosis, macropinocytosis, the clathrin-dependent pathway, the caveolin-dependent pathway and the clathrin- and caveolin-independent pathway are responsible for the majority of endocytocytic activities in the eukaryotic cell. Following endocytosis, the vesicles are transported to the early endosome. The cargoes that are destined for degradation then continue their journey to the lysosome, while others are either recycled back to the plasma membrane or the trans-Golgi network (Besterman and Low, 1983; Soldati and Schliwa, 2006; Mayor and Pagano, 2007; Alberts et al., 2008). 1.3 The biosynthetic/secretory pathway Secretion is an important biological activity of all eukaryotic cells by which they release a variety of substances, such as, hormones, antibodies, neutrotransmitters, extracellular matrix proteins and/or others into the environment. Proper functioning of the secretory pathway is important for the development and maintenance of normal physiology of an organism. A large number of eukaryotic proteins traverse the secretory pathway en route to their final destination either inside or outside the cell. These include proteins that are destined to be secreted from the cell, enzymes and other residents in the lumen of the endoplasmic recticulum (ER), the ER-Golgi intermediate compartment (ERGIC), the Golgi complex and the lysosome, as well as integral proteins in the membranes of these organelles and the plasma membrane (Alberts et al., 2008; Derby and Gleeson, 2007 Lodish et al., 2008). 1.3.1 Conventional secretory pathway Most of the secretory proteins are exported from cells through the conventional ERto-Golgi secretory pathway (Nickel and Rabouille; 2009). The majority of secretory proteins contain an amino-terminal signal peptide or internal signal sequences that direct their sorting to the ER. These proteins are usually co-translationally translocated into the ER (Osborne et al., 2005). After proper folding, the proteins exit the ER at specialized membrane domains called ER exit sites (ERES), from which cargo-containing coat protein complex II (COPII)-coated vesicles form. These newly synthesized proteins reach the Golgi apparatus and moves through the various cisternae of the Golgi, where they are post-translationally modified and processed (Lee et al., 2004; Tang et al., 2005; Derby and Gleeson, 2007; Nickel and Rabouille; 2009). Coat protein complex I (COPI)-coated vesicles are mainly formed in the Golgi and are involved in the retrograde transport of Golgi components back to the ER, intra-Golgi transport and anterograde trafficking of certain proteins (Rabouille and Klumperman, 2005). At the trans-Golgi network (TGN), the secretory proteins are sorted and dispatched to different subcellular destinations such as the endosome, lysosome and the plasma membrane by clathrin-coated vesicles (Gu et al., 2001; Gerdes, 2008). 1.3.2 Non-conventional secretory pathway Most of the extracellular proteins are secreted by means of the conventional ER-Golgi secretory pathway. However, a growing number of proteins that does not have the signal peptide have been demonstrated to be released via non-conventional secretory pathways independent of the ER and Golgi (Prudovsky et al., 2008; Nickel and Rabouille, 2009). The non-conventional protein release is resistant to Brefeldin A (BFA), which is a specific inhibitor of conventional secretion that acts by inhibiting protein transport from the ER to Golgi (Misumi et al., 1986). A large variety of products such as cholesterol, lipids, cytokines, enzymes, heat shock proteins, chromatin-associated proteins, viral proteins and many others were demonstrated to be released via the unconventional secretory pathway (Marie et al., 2008; Prudovsky et al., 2008; Nickel and Rabouille, 2009). Four general mechanisms are employed by non-classically secreted proteins to exit the cell: (i) Membrane blebbing: Secretory transglutaminase is concentrated at the cell membrane near where membrane blebs are formed. The protein is secreted when the blebs detached from the cell surface and ruptures in the extracellular environment (Aumuller et al., 1999). (ii) Endo-Lysosomal pathway: HSP70 was found to translocate from the cytosol into the endo-lysosomal compartments, which then fuse with the cell membrane to release their content into the exterior of the cell (Mambula and Calderwood, 2006). (iii) Exosome mediated secretion: Exosomes are small membrane vesicles (50–100 nm) that are secreted. They arise by the inward budding of the membrane into the lumen of endosome. As a result, cytosolic components were engulfed into the vesicles located inside the enlarged endosomes known as multivesicular bodies (MVBs). The endosome-derived MVBs fuse with the plasma membrane and release the exosomes which contains the cytosolic components into the extracellular environment (de Gassat et al., 2004). Heat shock protein 90 (Hsp90) was found to be exported through this mechanism (Yu et al., 2006). (iv) Translocation across the plasma membrane: Proteins could be secreted to the extracellular environment by translocating from the cytosol across the plasma membrane. Fibroblast Growth Factor (FGF1) was found to be in a complex with two other proteins, p40 Syt1 and S100A13 (LaVallee et al., 1998; Mouta Carreira et al., 1998). This association is required for the direct translocation of this protein complex across the plasma membrane into the extracellular environment. However, the machinery that mediates membrane translocation of this protein complex has yet to be identified (Marie et al., 2008; Prudovsky et al., 2008; Nickel and Rabouille, 2009). 1.4 Exiting the ER To begin their journey along the biosynthetic-secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond must first exit the ER. To earn the right of passage, the proteins have to undergo rigorous quality control checks to make sure that they are properly folded and assembled into their multimeric protein complexes. This quality-control step is very important, as misfolded or misassembled proteins could potentially interfere with the functions of normal proteins if they were transported onward. Misfolded intermediates are retained in the ER by interactions with chaperones, while terminally misfolded proteins are directed back into the cytosol for proteasome mediated degradation. Proteins that are correctly folded and assembled are then packaged and transported out of the ER (Gorelick and Shugrue, 2001; Ellgaard and Helenius, 2003; Mancias and Goldberg, 2005; Hughes and Stephens; 2008). The forward transport of proteins from the ER to the Golgi is mediated by Coat protein complex II (COPII), a membrane coat that forms ER-derived vesicles. A series of events must occur during the formation of COPII coated vesicles. COPII subunits must first be recruited to the correct site on the membrane of the ER exit sites (ERES). Transmembrane and soluble cargo proteins destined for export to the Golgi are then recruited and concentrated into the nascent COPII buds. Molecules that cycle between the ER and Golgi compartments are also incorporated into COPII vesicles. After the release of the ER-derived vesicles, the COPII coat is disassembled to allow vesicle fusion with the Golgi and the recycling of COPII components back to the ER (Barlowe, 1998; Tang et al., 2005; Lee and Miller, 2007; Sato and Nakano, 2007; Hughes and Stephens, 2008). 1.5 Recruitment of Cargo While some proteins are shown to exit the ER via a non-specific, inefficient process known as ‘‘bulk membrane flow’’, it is now clear that the majority of secretory cargo proteins are actively sorted from the ER resident proteins and incorporated into prebudding complexes. This selective cargo capture is mediated by the export signals found in the exposed cytoplasmic domain of the cargo. Export signals of most transmembrane cargo proteins are thought to be recognized by COPII subunits and are recruited into the pre-budding complexes directly. Some transmembrane and most soluble cargo binds indirectly to COPII through transmembrane cargo adaptors/receptors (Barlowe, 2003; Lee et al., 2004; Tang et al., 2005; Sato and Nakano, 2007; Hughes and Stephens, 2008). 1.5.1 Transmembrane cargo proteins Several classes of ER export signals have been identified on the cytoplasmic tails of transmembrane cargo proteins. The di-acidic motifs, [(D/E)-x-(D/E), where x represents any amino acid] was first described in the mammalian system for the vesicular stomatitis virus G protein (VSVG) (Nishimura et al;, 1997). Many other proteins such as mammalian potassium channel protein Kir2.1 (Ma et al., 2001), cystic fibrosis transmembrane conductance regulator (CFTR) (Wang et al., 2004), and yeast membrane proteins such as Sys1p and Gap1p (Votsmeier and Gallwitz, 2001) were also found to contain the di-acidic motifs at their cytoplasmic domain, which are required for their ER exit and surface expression. The di-acidic motifs on these cargoes mediate their interations with COPII subunits. Sys1p interacts with Sec23Sec24 via its di-acidic residues (Votsmeier and Gallwitz, 2001) and Gap1p requires its di-acidic motif to form pre-budding complexes with Sar1 and Sec23–Sec24 (Malkus et al., 2002). CFTR interacts with Sec23-Sec24 via its di-acidic motif. Mutation in the di-acidic motif greatly reduced its interaction with the Sec23-Sec24 complex and almost completely abolished its export from the ER (Wang et al., 2004). The di-hydrophobic/di-aromatic motifs (FF, YY, LL, or FY) contain a simple combination of two adjacent hydrophobic residues at or near the C-terminus of a protein. They are found in the cytoplasmic domains of several transmembrane cargos such as the Erv41/46p complex (Otte and Barlowe, 2002; Welsh et al., 2006), the p24 family proteins (Fiedler et al., 1996; Dominguez et al., 1998), and the ER-Golgi intermediate compartment (ERGIC)53/Emp47p family proteins (Kappeler et al., 1997; Sato and Nakano, 2002). Members of the p24 family contain the di- hydrophobic/aromatic motif that is present in the cytoplasmic domain. This domain was shown to participate in the binding of Sec23 and is required for its export from the ER. Mutational analysis of this domain impaired binding with Sec23 and caused redistribution of these proteins to the ER (Dominguez et al., 1998). ERGIC53 contains a conserved pair of aromatic residues (FF) at the extreme C-terminus that is necessary for transport from the ER (Kappeler et al., 1997), while its yeast homologues possess bulky hydrophobic residues at their C-termini (LL) that are required for export from the ER and proper localization (Nufer et al., 2002; Sato and Nakano, 2002). These terminal signals were shown to be able to bind COPII subunits (Kappeler et al., 1997; Nufer et al., 2002). Other bulky hydrophobic amino acids can substitute for this C terminal motif suggests some flexibility in this signal. It was also shown that when appropriately spaced from the membrane, the C-terminal valine residue is sufficient for efficient export from the ER (Nufer et al., 2002). SNAREs important for ER-Golgi transport were also packaged into the COPII coat buds. The yeast SNAREs Bet1p, Sed5p, and Sec22p all binds to Sec24p on different binding sites with different affinities. Structural and biochemical characterization of the cytoplasmic domains of these yeast SNARE proteins has identified the YxxxNPF and LxxME motifs on Sed5 and LxxLE motif on Bet1 as ER export signals (Mossessova et al., 2003). 1.5.2 Export of soluble cargo and membrane proteins without ER export motifs Export of soluble cargo proteins and glycosylphosphatidylinositol (GPI)-anchored membrane proteins that not present cytoplasmic signals requires specific transmembrane cargo receptors to mediate their interaction with the COPII proteins in the cytoplasm. The membrane proteins Erv29p, ERGIC53, the p24 proteins are examples of such transmembrane cargo receptors (Barlowe, 2003; Tang et al, 2005; Baines and Zhang, 2007). Erv29p, a multispanning membrane protein that cycles between the ER and the Golgi, is required for the efficient incorporation of glycoproα-factor (gpαf), the precursor of α-factor mating pheromone, into a COPII vesicle (Belden and Barlowe 2001). The hydrophobic motif (-I-L-V-) within the pro-region of gpαf is necessary for Erv29p binding and efficient packaging into COPII vesicles (Otte and Barlowe, 2004). Database searches revealed that other soluble secretory proteins also appear to possess such hydrophobic residues in similar positions, 10 implying similar mechanisms in cargo receptor interaction (Sato and Nakano, 2007). ERGIC53 is in fact a mannose-selective and calcium-dependent transmembrane lectin, (Appenzeller et al. 1999) that is required for the secretion of a number of glycoproteins including blood coagulation factors (Nichols et al., 1998), cathepsin C and cathepsin Z in mammalian cells (Appenzeller et al. 1999). The p24 proteins are type I transmembrane proteins of about 24 kDa in size, and contain a single membrane spanning region, a large lumenal domain, and a short cytoplasmic tail. They have been implicated in cargo protein sorting, COPI and COPII vesicle formation and regulation of vesicular transport in eukaryotic cells (Springer et al., 2000; Barlowe, 2003; Tang et al., 2005, Strating and Martens, 2009). In yeast, the putative yeast homolog of mammalian p24, Emp24p is essential for efficient packaging of glycosylphosphatidylinositol (GDI)-anchored protein Gas1p into COPII vesicles (Muniz et al., 2000). Members of this family may be required for essential functions in higher eukaryotes. Knockout of the p23 gene, a member of the p24 family, resulted in embryonic lethality (Denzel et al., 2000). In contrast a yeast strain that lacks all members of the p24 family is still viable (Springer et al., 2000). 1.5.3 COPII proteins and cargo recognition Several lines of evidence indicate that a family of paralogous Sec24 proteins functions in cargo recognition. In S. cerevisiae, there are at least two Sec24 isoforms, Lst1p and Iss1p (Roberg et al., 1999; Peng et al., 2000; Shimoni et al., 2000) In mammalian cells, there are isoforms of Sec23 and isoforms of Sec24 identified (Pagano et al., 1999; Bonifacino and Glick, 2004). The presence of multiple Sec23 and Sec24 isoforms appear to bring about combinatorial diversity to cargo selection for exported from the ER (Gurkan et al., 2006). In yeast, Lst1 is required for efficient 11 export of specific transmembrane cargoes from the ER (Roberg et al., 1999; Shimoni et al., 2000). The spectrum of cargo packaged into vesicles synthesized with Sec23/Lst1 was very distinct from those generated with Sec23/Sec24 (Miller et al., 2003). The observation that Sec23/Sec24 displays binding affinities for both di-acidic (Votsmeier and Gallwitz, 2000) and di-hydrophobic/aromatic motifs (Kappeler et al., 1997; Dominguez et al., 1998; Belden and Barlowe, 2001), also supports a direct role for Sec24 in cargo recognition. Sar1 might also contribute to cargo recognition through direct association with export signals. Sar1 was found to interact directly with the cytoplasmic tail of VSVG (Aridor et al., 2001). Interactions between Sar1 and other cargo have also been documented (Springer and Schekman, 1998; Belden and Barlowe, 2001; Otte and Barlowe, 2002). However, the affinity and specificity of these associations are not well characterized (Barlowe, 2003). 1.6 COPII coat assembly In Saccharomyces cerevisiae, protein export from the ER appears to proceed randomly throughout the ER membrane. However, in most other eukaryotes, protein export from the ER occurs at specialized regions known as transitional ER (tER) or ERES, which are distributed throughout the cell periphery, but concentrated at the perinuclear region (Orci et al., 1991; Bannkyh et al., 1996). Live cell imaging revealed that these sites are stable and long-lived structures. The cells appear to regulate the ERES sizes via de novo synthesis, fission and fusion of existing ERES (Stephens, 2003). COPII proteins are enriched at the ERES and mediate protein export from these sites. The COPII genes were first identified and characterized in a yeast genetic screen for mutants that are defective in secretion (Novick and Schekman, 1979; Novick et al., 1980). They are responsible for the accumulation of secretory cargo, the deformation of the membrane at the ERES and generation of subsequent 12 transport vesicles (Gurkan et al., 2006; Sato and Nakano, 2007; Hughes and Stephens, 2008). The COPII coat is formed through sequential binding of three cytosolic components, a small GTP binding protein, Sar1 (Nakano and Muramatsu, 1989; Barlowe et al., 1993), the Sec23/Sec24 heterodimer complex (Hicke et al., 1992) and the Sec13/Sec31 heterotetramer complex (Salama et al. 1993), respectively, to the ER membrane. The sequence of COPII protein assembly was established by the sequential addition of yeast COPII components to an in vitro assay. This order of assembly was subsequently confirmed in mammalian cells (Barlowe et al., 1994; Kuge et al., 1994; Aridor et al., 1995, Lee et al., 2004). Initiation of COPII coat assembly occurs upon the activation of Sar1, which is normally present in the cytosol as an inactive GDP-bound form (Nakano and Muramatsu, 1989; Barlowe et al., 1993). Upon loading with GTP, Sar1 (Sar1-GTP) is activated and is recruited to the ER membranes (Nakano and Muramatsu, 1989). Sar1 activation is facilitated by Sec12, an ER-resident guanine nucleotide exchange factor (GEF) (Barlowe and Schekman, 1993; Weissman et al., 2001). Activated Sar1 binds to the membrane and recruits the Sec23/Sec24 complex (Hicke and Schekman 1989; Kaiser and Schekman 1990; Hicke et al. 1992; Barlowe et al. 1994). Sec23 is a GTPase activating protein (GAP) for Sar1 (Yoshihisa et al., 1993), while Sec24 is required for cargo binding (Miller et al., 2003; Mossessova et al., 2003). Together with cargo, Sar1 and the Sec23/Sec24 heterodimer form the inner shell or the prebudding complex (Bi et al., 2002). The pre-budding complex then recruits the Sec13/Sec31 heterotetramer (Barlowe et al., 1994, Matsuoka et al., 1998) which constitutes the outer shell of the COPII coat (Bi et al., 2002). Recruitment of Sec31 further stimulates Sec23’s GTPase activating protein (GAP) activity towards Sar1 (Bi et al., 2007). Propagation of the Sec13/Sec31 scaffold into a cage structure may Sec12 S GDP Sar1‐GTP Sar1‐GTP Sec23/Sec24 Cargo Export Signals pre‐buddingg complex Sec13/Sec31 heterotetramer Polymerizattion buddingg © OYS’09 Figu ure 1.2. Selective uptake of cargo and COPII vesiclle formation. CO OPII vesicle formation is initiated uponn activation Sar1 bby the transmembraane guanine nucleootide exchange facctor, Sec12. Activaated Sar1-GTP binds to the ER membrrane and recruits thhe Sec23/Sec24 heterodimer, forming the pre-budding coomplex. Cargo is caaptured and concenntrated to the pre-bbudding complex. The pre-buddingg complexes are clustered by the Sec113/Sec31 subcomplex, generating COP PII vesicles. Adaptted from Sato and N Nakano, 2007. Sar1‐GDP ER lume en GTP Saar1‐GDP 13 14 function to both shape the membrane curvature as well as to concentrate pre-budding complexes into the nascent bud (Stagg et al., 2006; Fath et al., 2007; Hughes and Stephens, 2008). Hydrolysis of GTP to GDP by Sar1 disassembles the COPII coat from the budded vesicles, consequently allowing fusion of the vesicle to downstream acceptor compartments (Barlowe et al., 1994; Antonny et al., 2001). Figure 1.2 illustrates the sequence of COPII coat assembly. Even though COPII vesicle formation could be minimally reconstituted using purified Sec23/Sec24, Sec13/Sec31, and Sar1 locked into the activated conformation with a non-hydrolyzable GTP analogue (Matsuoka et al., 1998), additional regulatory factors are kown to contribute to the COPII assembly. In yeast, Sec16p, an ER-associated peripheral membrane protein is required for COPII budding in vivo (Espenshade et al., 1995; Supek et al., 2002). It is a large multi-domain protein which binds to Sec23p, Sec24p, and Sec31p (Espenshade et al., 1995; Gimeno et al., 1996; Shaywitz et al., 1997; Supek et al., 2002). Sed4 is an integral membrane protein located at the ER membrane. Its deletion retards transport from the ER to the Golgi. Sed4 shares significant homology with Sec12, but does not exhibit any GEF activity. It weakly inhibits the GTPase-activating (GAP) activity of Sec23p toward Sar1p and may function as an inhibitor of GTP hydrolysis by Sar1p (Gimeno et al., 1995; SaitoNakano and Nakano, 2000). 1.7 The COPII subunit proteins 1.7.1 Sar1- the small GTPase Sar1 (supressor of activated ras1) is a small G-protein of the Ras superfamily that is the master regulator of COPII vesicle biogenesis (Nakano and Muramatsu, 1998). 15 Sar1, like other small G-proteins, utilizes guanine nucleotide exchange to switch between activate and inactive states (Kirk and Ward, 2007). Upon activation, Sar1 undergoes a conformational change and embeds its N-terminal helix into the ER membrane (Huang et al., 2001). On top of its role as a membrane anchor, Sar1 has been found to play a role in stimulating curvature of the membrane and vesicle fission. Addition of activated Sar1 to synthetic liposomes led to deformation of spherical liposomes into elongated thin tubules (Bielli et al., 2005; Lee et al., 2005). This deformation process results from the intercalation of the Sar1 N-terminal helix into the lipid bilayer (Lee et al., 2005). Addition of wild type Sar1 together with high concentrations of Sec23/Sec24 and Sec13/Sec31 complex to synthetic liposomes generated free vesicles, while the addition of Sar1 lacking the helical domain yielded numerous coated buds that are unable to fully detach from the membrane (Lee et al., 2005). Sar1 locked into its active form with a non-hydrolysable GTP, or a Sar1 mutant deficient in GTP hydrolysis, inhibited vesicle release in an in vitro budding reaction (Bielli et al., 2005). These observations suggest that Sar1 is required to initiate membrane deformation, and that its GTPase activity is essential for efficient vesicle fission (Bielli et al., 2005; Lee et al., 2005). However, exactly how Sar1 promotes vesicle fission is not known. 1.7.2 Sec23/Sec24 complex The membrane recruitment and activation of Sar1 is followed by the binding of cytosolic Sec23/Sec24 complex. Sec23/Sec24 functions as an adaptor platform for cargo selection during COPII vesicle budding (Gurkan et al., 2006). It also recruits the Sec13/Sec31 heterotetramer to the pre-budding complex (Barlowe et al., 1994, Matsuoka et al., 1998). The structure of Sec23/Sec24 in complex with Sar1 has been solved. Structural analysis of the Sec23/Sec24 heterodimer revealed a curved bowtie- 16 shaped complex with a concave surface for binding the membrane vesicle. The interface between Sec23 and Sar1 is stabilized by the bound GTP molecule. Sec23 and Sec24 were found to be structurally homologous to each other and contain similar domain organisations (Bi et al., 2002). They each comprise of an α-helical region, a β-barrel region, a zinc-finger domain, a gelsolin-like domain and a trunk domain. The Sec23/Sec24 heterodimers were formed through interactions between their respective trunk domains (Bi et al., 2002; Bickford et al., 2004). The zinc-finger domain in Sec24 forms cargo selection domain that binds to ER export signals containing acidic residues. The β-barrel domains forms part of the platform that creates the concave inner surface, and the α-helical region in Sec23 contributes residues that contact Sar1 and the gelosin domains may constitute the site for Sec13/Sec31 interaction. The gelosin domain of Sec23 also contributes to the interaction with Sar1 through a crucial arginine residue (R722) that is required for the GTPase activating activity of Sec23 (Bi et al., 2002). The concave “inner” surface of the Sar1-Sec23/Sec24 complex, enriched in basic amino acids, has a net positive charge and contacts the membrane. This affinity of Sec23/Sec24 for acidic phospholipids may also contribute to the membrane deformation that is a prerequisite for vesicle formation (Bi et al., 2002; Bickford et al., 2004). Even though Sec23 and Sec24 have striking structural similarities, and are unique in their functions (Bi et al., 2002). Sec23 is a Sar1-specific GAP (Yoshihisa et al., 1993), while Sec24 functions as a cargo adaptor platform that is responsible for the efficient recruitment of specific cargo proteins that are destined for ER export (Miller et al., 2003; Mossessova et al., 2003). Subsequent mutagenesis and structural analysis have revealed three independent cargo-binding sites on the membrane-facing surface of various Sec24 isoforms. They are termed the A-, B- and C-sites 17 respectively (Miller et al., 2003; Mossessova et al., 2003). The A-site comprises a hydrophobic pocket that recognises a YxxxNPF motif on the Sed5, a Golgi syntaxin homolog (Mossessova et al., 2003). The di-acidic motifs are recognised by the B-site, which is formed by the zinc-finger domain. COPII vesicles generated with Sec24 that was mutated in the B-site showed defects in capturing the cargo proteins tested. The proteins affected by this mutation contains diverse ER export signals suggest that the B-site has the capacity to recognize a variety of signals. Mutation of the C-site results in defects in the capture of the SNARE molecule, Sec22, in vitro. Since packaging of Sec22 was also impaired by mutation of the B-site, and Sec22 likely binds to Sec24 in a complex manner (Miller et al., 2003). 1.7.3 Sec13/Sec31 complex There are two mammalian Sec31 isoforms, Sec31A (~135 kDa) and Sec31B (~129 kDa) (Shugrue et al., 1999; Tang et al., 2000; Stankewich et al., 2006). Currently, only one isoform has been detected in S. cerevisiae. At present, the only known function of Sec31 is restricted to the COPII machinery and ER export (Gurkan et al., 2006). Similar to yeast, there are two mammalian Sec13 isoforms, Sec13-like-1 and the larger Sec13-like. The larger isoform SEC13-like, does not have a role in COPII function in both mammals and yeast (Swaroop et al., 1994; Shaywitz et al., 1995; Siniossoglou et al., 1996; Tang et al., 1997). Sec13 was also found in the nuclear pore complex and the mitotic spindle (Siniossoglou et al., 1996; Loiodice et al., 2004). These observations suggest that Sec13 function is not limited to the COPII machinery. The Sec13/Sec31 complex is the last to be recruited to the COPII coat and does not contact the membrane directly (Matsuoka et al., 2001). The propagation of the Sec13/Sec31 lattice into a cage structure may therefore function to shape the membrane curvature initiated by Sar1–Sec23/Sec24 and to concentrate Sec23/Sec24– 18 cargo complexes into the nascent bud (Stagg et al., 2006; Fath et al., 2007; Lee and Miller, 2007; Hughes and Stephens, 2008). Sequence analysis of Sec13 and Sec31 indicated the presence of WD40 repeats. WD40 repeats are short repeated sequences of about 40 amino acids, named after the presence of a conserved Tryptophan (W) and Aspartic acid (D) motif (Neer et al., 1994). The WD40-repeat domains are speculated to form a circularized β-propeller structure (Li and Roberts, 2001). Sec31 contains WD40 repeats at its N terminus, with the rest of the protein composed of two regions of α-solenoid structure that are separated by a region of low complexity, while Sec13 is almost entirely composed of WD40 repeats (Gurkan et al., 2006; Fath et al., 2007). In vivo, Sec13 and Sec31 form a stable heterotetramer in the cytosol composed of two molecules each of Sec13 and Sec31 (Salama et al., 1993; Salama et al., 1997; Shugrue et al., 1999; Tang et al., 2000; Lederkremer, et al, 2001) via their WD40 domains (Devos et al., 2004; Gurkan et al, 2006; Stagg et al., 2006; Fath et al., 2007). Cumulative data from biochemical and electron microscopy studies indicated that the heterotetramer complex is arranged as an elongated globular-domain structure with two-fold symmetry in the order of Sec31–Sec13–Sec13–Sec31 (Lederkremer et al., 2001; Matsuoka et al., 2001; Stagg et al., 2006; Fath et al., 2007). In the Sec13/Sec31 assembly unit, the N- terminal of each Sec31 polypeptide folds back on itself, generating an interlocked dimer. The Sec13 molecule binds to residues 380-406 of Sec31, separating the β-propeller structures from the α-solenoid structures of Sec31 (Fath et al., 2007). Under certain ionic conditions, twenty-four copies of the Sec13/Sec31 assembly unit can selfassemble into a cuboctahedron1-like structure of about 60 nM in diameter, similar to A cuboctahedron is a polyhedron with eight triangular faces and six square faces. It has 12 identical vertices, with two triangles and two squares meeting at each, and 24 identical edges, each separating a triangle from a square (Williams, Robert (1979). The Geometrical Foundation of Natural Structure: A Source Book of Design. Dover Publications, Inc. ISBN 0-486-23729-X) 19 the diameter of the COPII vesicles in vivo (Stagg et al., 2006). The β-propeller domain of Sec31, positioned at the ends of the assembly unit mediates the vertex contacts that propagate the COPII cage (Stagg et al., 2006; Fath et al., 2007; Lee and Miller, 2007). The molecular structure of the complete COPII coat containing both Sec13/Sec31 and Sec23/Sec24 complexes was recently solved using cryo-electron microscopy (Stagg et al., 2008). Purified components of the two major COPII complexes, the Sec13/Sec31 complex and the Sec23/Sec24 complex were allowed to self-assemble in solution. The self-assembled COPII cage exhibited the structure of an icosidodecahedron2 and appears to have three layers. The outer layer corresponds to the Sec13/Sec31 cage followed by the middle layer which is made up of the Sec23/Sec24 complex. A regular complex consisting of four dimers of the Sec23/Sec24 complex were positioned beneath the vertices and the edges of the Sec13/Sec31 lattice. The innermost layer lacks regular structure and was suggested to be the unassembled Sec13/Sec31 and/or the Sec23/Sec24 complexes (Stagg et al., 2008). Results from crystal structures and density maps of the COPII proteins revealed a flexible hinge region at the vertices of the Sec13/Sec31 scaffold, which can direct geometric cage expansion to accommodate a wide range of large and bulky cargo (Stagg et al., 2006; Lee and Miller, 2007; Stagg et al., 2008). An icosidodecahedron is a polyhedron with twenty triangular faces and twelve pentagonal faces. It has 30 identical vertices, with two triangles and two pentagons meeting at each, and 60 identical edges, each separating a triangle from a pentagon (Williams, Robert (1979). The Geometrical Foundation of Natural Structure: A Source Book of Design. Dover Publications, Inc. ISBN 0-486-23729-X). 20 1.7.4 Sec12 Sec12 functions as the GEF for Sar1. It is a type II transmembrane ER resident protein with a large cytosolic domain that promotes Sar1 activation (Barlowe and Schekman, 1993; Weissman et al., 2001). WD40 repeats were detected in the cytosolic domain of Sec12 (Chardin and Callebaut, 2002). Although there is currently no detailed structural information on the Sec12-Sar1 interface, the WD40 repeats are believed to mediate Sar1 interaction as mutagenesis of several residues in the WD40 repeats disrupted Sar1 binding and nucleotide exchange activity (Chardin and Callebaut, 2002). As Sec12 is a membrane protein and serves to activate Sar1, it may be one of the most upstream components of the COPII budding process (Tang et al., 2005), and may be involved in the nucleation of the ERES. Mammalian and S. cerevisiae Sec12 was distributed throughout the ER membranes (Nishikawa and Nakano, 1993; Weissman et al., 2001; Stephens, 2003), while Sec12 in Pichia pastoris is localised to ERES puncta (Rossanese et al., 1999). Although overexpression of Sec12 in P. pastoris causes its localisation to the entire ER, it did not affect the distribution of downstream COPII components or Golgi localisation (Soderholm et al., 2004). Mammalian cells treated with Sec12 RNAi also did not exhibit obvious disruptions to ERES distribution and organisation, however, COPII regulated cargo export out of the ER was affected (Bhattacharyya and Glick, 2007). This indicates that Sec12 is needed for cargo transport out of the ER, but is not essential for organisation of the ERES. 1.7.5 Sec16 Sec16 was first identified in yeast as an essential component for secretion (Novick and Schekman, 1979; Novick et al., 1980). Sec16, a 240 kDa peripheral membrane protein associated with the ER, is required for ER-Golgi transport (Espenshade et al., 21 1995) and interacts with multiple components of the COPII machinery, including Sec23, Sec24, Sec31 and Sar1 (Espenshade et al., 1995; Gimeno et al., 1995; Gimeno et al., 1996; Shaywitz et al., 1997; Supek et al., 2002). It has been suggested that Sec16 serves to nucleate and/or regulate COPII vesicle formation (Shaywitz et al., 1997; Supek et al., 2002). The mammalian Sec16 homologues, Sec16L and Sec16S were recently identified (Watson et al., 2006; Bhattacharyya and Glick, 2007; Iinuma et al., 2007). The C-terminal domain of yeast Sec16 that interacts with Sec23 was present in Sec16L but not in Sec16S, suggesting that Sec16L is an orthologue of yeast Sec16, and the smaller homologue, Sec16S appears to confer a non-redundant function similar to the large protein (Bhattacharyya and Glick, 2007). Sec16 localizes with other COPII components to the ERES (Watson et al., 2006; Bhattacharyya and Glick, 2007; Iinuma et al., 2007). Overexpression of Sec16 resulted in a loss of the punctate localisation of the Sec23/Sec24 or Sec13/Sec31 complex, a reduction in peripheral ERGIC53 and β-COP puncta, an inhibition of VSVG exit from the ER and an altered Golgi morphology. Sec16 depletion mediated by RNA interference (RNAi) greatly reduced VSVG transport to the plasma membrane or the export of a Golgi protein following BFA washout. These results suggest that Sec16 is essential for ER export and ERES organisation (Watson et al., 2006; Bhattacharyya and Glick, 2007; Iinuma et al., 2007). 1.8 p125A p125A was first described as a Sec23 interacting protein at the ERES. It is a 125 kDa peripheral membrane protein with phopholipase A1 homology (Tani et al., 1999; Shimoi et al., 2005). The mammalian genome contains a paralogue, p125B which shares a remarkable sequence similarity with p125A throughout the entire sequence, but lacked the proline-rich N-terminal domain that is required for Sec23 interaction. 22 In vitro binding analysis showed that p125B does not interact with Sec23. The function of p125B is unclear. However, it was predicted to be a new member of the phosphatidic acid preferring phospholipsae A1 family that has a different cellular function to p125A (Nakajima et al., 2002). p125A was recruited to the ERES in an active Sar1p dependent manner. Activation of Sar1 enhanced the binding of p125A to membranes. Overexpression of p125A causes ERES clustering at the perinuclear region with downstream defects in ERGIC and Golgi protein localisation (Tani et al., 1999; Shimoi et al., 2005). p125A silencing using siRNA resulted in a loss of perinuclear accumulation of ERES but the peripheral sites were not affected. However, no defect in cargo transport was observed in p125A depleted cells. These results suggest that p125A might be involved in the architecture of ERES. As lower eukaryotic organisms such as yeast not contain p125A, p125A may play an important role in cellular processes that are unique to multicellular organisms. There are accumulating data that suggest the importance of phospholipids in vesicular transport (De Camilli et al., 1996; Blumental-Perry et al., 2006). Phosphatidylinositol-4-phosphate (PtsIns4P) is required to for COPII-mediated ER export. Biochemical and morphological studies in vitro revealed localized and dynamic formation of PtsIns4P at the ERES. PtsIns4P is also essential for Sar1induced COPII nucleation at the ERES. Although p125A exhibit significant sequence homology to phospholipase A1, it did not have any detectable phospholipase activity (Nakajima et al., 2002). However, p125A was found to bind phosphatidylinositol phosphate (Iinuma et al., 2007), and may play a role in anchoring COPII proteins to the ERES. 23 1.9 Rationale of study The first mechanistic understanding of the function of the various components of COPII pertaining to vesicle formation came from the yeast system (Kuehn et al., 1998). We now have a fairly good understanding of some of the molecular and structural interactions that drive COPII assembly (Tang et al., 2001). Homo sapiens express about five times more proteins (~30,000) compared to Saccharomyces cerevisiae (~6300) (Alberts et al., 2008). For each of the yeast COPII genes identified, there are at least or more isoforms in mammals (Barlowe, 2003). The presence of multiple isoforms brings about combinatorial diversity for COPII cage formation in mammals, indicates a greater complexity in the regulation of COPII- mediated protein transport. Therefore, even though yeast proteins have provided meaningful insights to this process, they are unable to provide a comprehensive picture for the mammalian system. The Sec13/Sec31 complex, is one of the last protein complexes to be recruited onto membranes prior to vesicle formation, and may be linked to regulatory mechanisms that govern ER exit. Tang et al., 2000, demonstrated that rat liver cytosol depleted of proteins potentially interacting with the C-terminal fragment of Sec31A, a COPII coat protein, was unable to support ER to Golgi transport of VSVG in a semi-intact cell system. This indicates that a cytosolic factor(s) sequestered by the C-terminal fragment of Sec31A is essential for the function of endogenous of Sec31A in ER export of VSVG. Since not much was known about the interacting partners of the mammalian Sec13/Sec31 complex, identification of Sec13/Sec31 interacting proteins that are involved in ER-to-Golgi transport may further elucidate underlying regulatory mechanism of COPII mediated ER export in mammals. 24 In this project, p125A, a Sec23 interacting protein (Tani et al., 1999), was identified as a Sec31A interacting protein using GST-pulldown and mass spectrometry. Deletion mutants of p125A were analyzed for their binding affinities towards Sec31A. Morphological and biochemical techniques were employed to study the effects of p125A gene silencing on ERES and Golgi morphology, and cargo export from the ER. Findings from these analyses are detailed in Chapter 3. [...]... Sar1 (Sar1-GTP) is activated and is recruited to the ER membranes (Nakano and Muramatsu, 19 89) Sar1 activation is facilitated by Sec12, an ER-resident guanine nucleotide exchange factor (GEF) (Barlowe and Schekman, 19 93; Weissman et al., 20 01) Activated Sar1 binds to the membrane and recruits the Sec23/Sec24 complex (Hicke and Schekman 19 89; Kaiser and Schekman 19 90; Hicke et al 19 92; Barlowe et al... 2000) 1. 7 The COPII subunit proteins 1. 7 .1 Sar1- the small GTPase Sar1 (supressor of activated ras1) is a small G -protein of the Ras superfamily that is the master regulator of COPII vesicle biogenesis (Nakano and Muramatsu, 19 98) 15 Sar1, like other small G-proteins, utilizes guanine nucleotide exchange to switch between activate and inactive states (Kirk and Ward, 2007) Upon activation, Sar1 undergoes... 2006; Bhattacharyya and Glick, 2007; Iinuma et al., 2007) 1. 8 p12 5A p12 5A was first described as a Sec23 interacting protein at the ERES It is a 12 5 kDa peripheral membrane protein with phopholipase A1 homology (Tani et al., 19 99; Shimoi et al., 2005) The mammalian genome contains a paralogue, p125B which shares a remarkable sequence similarity with p12 5A throughout the entire sequence, but lacked the... to the COPII machinery and ER export (Gurkan et al., 2006) Similar to yeast, there are two mammalian Sec13 isoforms, Sec13-like -1 and the larger Sec13-like The larger isoform SEC13-like, does not have a role in COPII function in both mammals and yeast (Swaroop et al., 19 94; Shaywitz et al., 19 95; Siniossoglou et al., 19 96; Tang et al., 19 97) Sec13 was also found in the nuclear pore complex and the... of yeast COPII components to an in vitro assay This order of assembly was subsequently confirmed in mammalian cells (Barlowe et al., 19 94; Kuge et al., 19 94; Aridor et al., 19 95, Lee et al., 2004) Initiation of COPII coat assembly occurs upon the activation of Sar1, which is normally present in the cytosol as an inactive GDP-bound form (Nakano and Muramatsu, 19 89; Barlowe et al., 19 93) Upon loading... 0-486-23729-X) 20 1. 7.4 Sec12 Sec12 functions as the GEF for Sar1 It is a type II transmembrane ER resident protein with a large cytosolic domain that promotes Sar1 activation (Barlowe and Schekman, 19 93; Weissman et al., 20 01) WD40 repeats were detected in the cytosolic domain of Sec12 (Chardin and Callebaut, 2002) Although there is currently no detailed structural information on the Sec12-Sar1 interface, the... repeats are believed to mediate Sar1 interaction as mutagenesis of several residues in the WD40 repeats disrupted Sar1 binding and nucleotide exchange activity (Chardin and Callebaut, 2002) As Sec12 is a membrane protein and serves to activate Sar1, it may be one of the most upstream components of the COPII budding process (Tang et al., 2005), and may be involved in the nucleation of the ERES Mammalian... and Sec 31 form a stable heterotetramer in the cytosol composed of two molecules each of Sec13 and Sec 31 (Salama et al., 19 93; Salama et al., 19 97; Shugrue et al., 19 99; Tang et al., 2000; Lederkremer, et al, 20 01) via their WD40 domains (Devos et al., 2004; Gurkan et al, 2006; Stagg et al., 2006; Fath et al., 2007) Cumulative data from biochemical and electron microscopy studies indicated that the heterotetramer... GTP analogue (Matsuoka et al., 19 98), additional regulatory factors are kown to contribute to the COPII assembly In yeast, Sec16p, an ER-associated peripheral membrane protein is required for COPII budding in vivo (Espenshade et al., 19 95; Supek et al., 2002) It is a large multi-domain protein which binds to Sec23p, Sec24p, and Sec31p (Espenshade et al., 19 95; Gimeno et al., 19 96; Shaywitz et al., 19 97;... et al., 2002) It has been suggested that Sec16 serves to nucleate and/or regulate COPII vesicle formation (Shaywitz et al., 19 97; Supek et al., 2002) The mammalian Sec16 homologues, Sec16L and Sec16S were recently identified (Watson et al., 2006; Bhattacharyya and Glick, 2007; Iinuma et al., 2007) The C-terminal domain of yeast Sec16 that interacts with Sec23 was present in Sec16L but not in Sec16S, . Sar1p and may function as an inhibitor of GTP hydrolysis by Sar1p (Gimeno et al., 19 95; Saito- Nakano and Nakano, 2000). 1. 7 The COPII subunit proteins 1. 7 .1 Sar1- the small GTPase Sar1. GTP, Sar1 (Sar1-GTP) is activated and is recruited to the ER membranes (Nakano and Muramatsu, 19 89). Sar1 activation is facilitated by Sec12, an ER-resident guanine nucleotide exchange factor. Sar1 (supressor of activated ras1) is a small G -protein of the Ras superfamily that is the master regulator of COPII vesicle biogenesis (Nakano and Muramatsu, 19 98). 15 Sar1, like other small