120 CHAPTER General discussion and future perspectives A SMART domain search analysis (http://smart.embl-heidelberg.de/) of p125A revealed a proline rich region at the N-terminus (142-259) (Tani et al., 1999), a WWE domain (261-342), a sterile alpha motif (SAM) (641-706) and a DDHD domain (771989) at the C-terminus. About 700 amino acids of the C-terminal region exhibit sequence similarity to phosphatidic acid-preferring phospholipase A1 and a consensus sequence found in most lipases is located at residues 581-585 (Gly-X-His-Ser-Leu, where X represent any amino acid) (Tani et al., 1999). However, p125A does not have any detectable phospholipase activity (Nakajima et al., 2002). A schematic illustration of p125A is shown in Figure 5.1. Other than the proline rich region, which was found to be involved in Sec23A interaction (Tani et al., 1999; Mizoguchi et al., 2000), and the Sec31A binding region that falls between residues 260-600 (current work), the functions of the other domains of p125A are still unclear. Figure 5.1. A schematic illustration of p125A. 121 5.1 The WWE domain The WWE domain, named after three of its conserved residues, tryptophan and glutamic acid and has been identified in diverse proteins with predicted ubiquitin- and ADP-ribosylation-related functions. This domain was thought to mediate proteinprotein interactions of these proteins (Aravind, 2001). The WWE domain was detected in many proteins with predicted ubiquitin-function. It is generally believed that ubiquitin and other ubiquitin-like proteins function as post-translational modifiers that regulate the function, interaction, localization and degradation of their target proteins (Welchman et al., 2005). Atg8, an ubiquitin-like protein is conjugated to the lipid phosphatidylethanolamine (PE) by an ubiquitin-like system. Atg8–PE mediates membrane tethering and hemifusion of the phagophore, and is required for the formation of autophagosomes (Nagatogawa et al., 2007). Atg8-PE is postulated to be a scaffold protein that supports membrane expansion and also have functions in cargo selection (Geng and Klionsky, 2008). Therefore, the presence of the WWE domain in p125A suggests that it may possess ubiquitin related functions and may associate with ubiquitin or ubiquitin related proteins. These interactions may mediate the degradation of the p125A-Sec13/31 complex, or alternatively, serve as a protein interaction platform to recruit ubiquitin-like proteins similar to Atg8, with functions in cargo selection, membrane tethering and fusion. It was also reported that the WWE domain of Deltex (a cytosolic effector of Notch signalling) mediates the formation of a heterodimer of Deltex with the ankyrin repeat domain of the Notch receptor (Zweifel et al., 2005), suggesting the potential capability of p125A to interact with proteins that contains ankyrin repeat domains. The ankyrin repeat is one of the most common protein-protein interaction motifs. 122 They are repeats of about 30-34 amino acids and occur in a large number of functionally diverse eukaryotic proteins (De Matteis and Morrow, 1998; Mosavi et al., 2004; Li et al., 2006). It was recently shown that the ankyrin binding capability of α1-Na+-K+-ATPase is required for its entry into the secretory pathway. Deletion of its ankyrin binding domain (MAB – minimal ankryin binding) impairs the transport of α1-Na+-K+-ATPase to the plasma membrane. Replacing the cytoplasmic domain of VSVG with the MAB from α1-Na+-K+-ATPase confers ankyrin dependency on the ER to Golgi trafficking to this VSVG-MAB chimera. Small hairpin RNA-mediated knockdown of ankyrin R in MDCK cells, trafficking of VSVG-MAB was impaired and was retained in the ER. Overexpression of a dominant negative mutant of Sar1a which inhibited the export of VSVG-MAB suggests that a COPII dependent mechanism is important for the ankyrin dependent export of the α1-Na+-K+-ATPase (Stabach et al., 2008). Ankyrin also functions as an adapter protein that links spectrin to membrane proteins (Beck and Nelson, 1998; De Matteis and Morrow, 1998; De Matteis and Morrow, 2000). The identification of Golgi associated ankryins and spectrins led to the discovery of the existence of a Golgi-localized membrane cytoskeleton (Beck et al., 1994; Beck et al., 1997). This spectrin/ankyrin skeleton contributes to the maintenance of Golgi structure and is important for efficient protein trafficking in the early secretory pathway. Spectrin links membranes to the motor proteins, as well as to all major filament systems (Beck and Nelson, 1998; De Matteis and Morrow, 1998; De Matteis and Morrow, 2000). A Golgi specific spectrin, βIII spectrin, was demonstrated to tether vesicles to the dynein-dynactin complex (Holleran et al., 2001), thereby enabling the transport of organelles and vesicles along microtubules. 123 The findings by Stabach et al. (2008), on α1-Na+-K+-ATPase export demonstrated a novel way of protein export facilitated by ankyrin, which is COPII dependent. However, the exact mechanism is still not clear. The ankyrin binding sequences may function as an export signal for incorporation into the export vesicles. Ankyrin also provides a connection for transport vesicles with the microtubules. Ankyrin interacting proteins associated with COPII may be key players for such modes of cargo recognition and protein export. 5.2 The DDHD domain The DDHD domain is about 180 residues long and contains four conserved DDHD (Asp-Asp-His-Asp) residues by which it was named. This four conserved residues may form a metal binding site and this pattern of conservation of metal binding residues is often observed in phosphoesterase catalytic domains. The DDHD domain is found in a family of phospholipases as well as the Nir/rdgB group of proteins (Lev et al., 1999). Phospholipases are implicated in the regulation of lipid transport and metabolism, intracellular trafficking, secretion, and vesicular transport (Brown et al., 2003). Drosophila retinal degeneration B (rdgB) is the first of the Nir/rdgB family to be identified. It plays an essential role in photoreceptor membrane renewal and biogenesis. Other proteins of this family are structurally and functionally related to the Drosophila homolog and have been implicated in trafficking, metabolism and signalling of lipids and as well as the regulation of cytoskeletal elements. The exact function of this domain is not clear at present (Lev, 2004). It is however of interest to note that metal ions such as calcium have been implicated in various stages of vesicular traffic between the ER and the Golgi (Beckers and Balch, 1989; Pind et al., 1994; Ivessa et al., 1995; Ahluwalia et al., 2001; Chen et al., 2002, Hasdemir et al., 2005). 124 Apoptosis-linked gene (ALG-2) was recently demonstrated to localize to the ERES via a calcium-dependent interaction with Sec31A, and is required for stabilizing Sec31A’s association with the ERES (Yamasaki et al., 2006). The DDHD domain in p125A may therefore contain a putative metal ion binding site, which may function to regulate p125A’s protein interactions and stability of interactions in a metal ion dependent manner similar to ALG-2. 5.3 The Sterile Alpha Motif The SAM (Sterile Alpha Motif) domain is an evolutionarily conserved domain of about 65-70 amino acids. It was first identified in yeast proteins that are essential for sexual differentiation. The SAM domain is also found in a myriad of signaling and nuclear proteins, and is involved in the regulation of many developmental processes (Ponting, 1995; Stapleton et al., 1999). Being able to homo- and heteroligomerise with other SAM domains (Schultz et al., 1996), and also to bind various non-SAM domain-containing proteins (Peterson et al., 1997), it can function as a protein-protein interaction module. The SAM domain could also serve as a platform for signal transduction events. The conserved tyrosine in the SAM domain, which could be phosphorylated may be binding sites for proteins containing phospho-tyrosine binding domains such as SH2 (Src Homology 2) (Russel et al., 1992). For example, for the EphB1 receptor tyrosine kinase, the conserved phospho-tyrosine in the SAM domain creates a binding site for the low molecular weight phosphotyrosine phosphatase (LMPTP) and a SH2 containing adapter protein, Grb10 (Thanos et al., 1999). In our study, both HA and myc tagged p125A were able to co-immunoprecipitate each other (Figure 3.10). The co-immunoprecipitation of myc-p125AFL by HA-p125AFL may occur indirectly via 125 interactions with the Sec13/31 heterotetramer complex, or directly with each other through their SAM domains. Binding experiments using purified proteins would be required to ascertain this possibility. 5.4 p125A as a scaffold platform for protein-protein interaction and regulation of COPII assembly and function One mechanism by which COPII vesicle biogenesis may be regulated is by posttranslational modification of the coat components. There is considerable evidence for protein kinases regulating membrane traffic at the ER-Golgi interface. The non-selective kinase inhibitor H-89 inhibits ER to Golgi transport by blocking the recruitment of Sar1 to the ER membrane, thus preventing the assembly of the COPII coated ERES (Aridor and Balch, 2000; Lee and Linstedt, 2000). Protein kinase A (PKA) was shown to regulate ER to the Golgi transport (Muniz et al., 1996; Lee and Linstedt, 2000), as well as the recycling of the KDEL-receptor from the early Golgi (Cabrera et al., 2003). The PCTAIRE kinases belong to the family of cyclin- dependent kinases and were regulated by PKA (Graeser et al., 2002). They can interact with Sec23 and were also shown to modulate ER to Golgi traffic (Palmer et al., 2005). Yeast Sec31 is a phosphoprotein, however, its specific site of phosphorylation has not been identified. Phosphorylation of Sec31 is required for its efficient function. Purified Sec31 that had been treated with phosphatase showed a 50% decrease in in vitro budding efficiency (Salama et al., 1997). Although the significance of Sec31 phosphorylation at the molecular level is unclear, this suggests that Sec31’s function may be regulated by kinases and phosphatases. Other modes of post-translational modifications such as ubiquitination and glycosylation have been implicated in regulating COPII activity. Sec23’s stability 126 and function appear to be also regulated by ubiquitin. This ubiquinitated form of Sec23 does not bind Sec24. Defects in de-ubiquitination results in the accumulation of the ubiquitinated Sec23 which is rapidly degraded, thereby impairing anterograde transport (Cohen et al., 2003). It was suggested that ubiquitination may serve more than targeting Sec23 for degradation. The attachment of ubiquitin to Sec23 might force the Sec23/24 subunits apart to facilitate coat disassembly prior to membrane fusion or to allow reassembly with different Sec24 isoforms (Lee and Miller, 2007). The transient inhibition of ER-to-Golgi transport in mammalian cells during mitosis is modulated by the glycosylation and phosphorylation state of Sec24. The interphase form of Sec24 was found to be O-glycosylated. Upon entry into mitosis, it is deglycosylated and phosphorylated concurrently. This mitotic form of Sec24 is unable to bind membranes, and is likely to be the basis of ER export inhibition during mitosis (Dudognon et al., 2004) Protein export from the ER is a highly regulated process. COPII proteins are regulated by a myriad of interactions with different proteins. We now have a comprehensive understanding of some of the molecular interactions that drive COPII assembly. However, many questions still remain, especially with regards to the regulation of vesicle biogenesis, and the flexibility of this process to encompass the vast diversity of cargo. The identification of COPII interacting proteins with roles in signal transduction, cell cycle and other regulatory pathways, may thus allow further dissections of the complex processes of the early secretory pathway. The various domains of p125A are implicated in protein-protein interactions, signal transduction processes and metal ion binding. The complexity of p125A’s domain structure thus provides a possible platform to recruit proteins that regulate COPII. It would 127 therefore be of interest in the immediate future to search for interacting partners of p125A that may be regulators or effectors of its function. 5.5 Conclusion In conclusion, this study demonstrated that p125A binds to Sec31A, and is in a ternary complex with the Sec13/31 heterotetramer in the cytosol prior to its recruitment to the membrane. p125A is localised to the ERES through its interaction with Sec31A. Depletion of p125A delays protein export out of the ER. It is therefore likely to play a role in stabilising COPII coat assembly, and may also function as a protein scaffold for the recruitment of cargo and/or signalling proteins that in the regulation of cargo exit from the ERES, . domain of about 65- 70 amino acids. It was first identified in yeast proteins that are essential for sexual differentiation. The SAM domain is also found in a myriad of signaling and nuclear proteins,. function as post-translational modifiers that regulate the function, interaction, localization and degradation of their target proteins (Welchman et al., 20 05) . Atg8, an ubiquitin-like protein. Sec3 1A s association with the ERES (Yamasaki et al., 2006). The DDHD domain in p12 5A may therefore contain a putative metal ion binding site, which may function to regulate p12 5A s protein