**3. Small GTPases as regulators of exocytosis**

While the exocyst complex has a clear role in exocytosis, the factors promoting the final orchestration of exocytosis are yet to be characterized. Emerging data highlights that small GTPases of the Ras super-family, including the Ras homologous (Rho), Ras-associated binding proteins (Rabs), adenosine ribosylation factors (Arfs), and Ras-like proteins (Ral) subfamilies, are involved in regulating distinct steps during exocytosis, some of which are mediated via interaction with the exocyst (reviewed in (Csepanyi-Komi, *et al*., 2011; Hutagalung & Novick, 2011; Segev, 2011)). Thus, there appears to be stage-specific requirements for small GTPase subfamily members during exocytosis (Figure 1).

The unique feature of the small GTPase superfamily (G-protein family) is the presence of a 20 kDa, catalytic domain (Bourne, *et al*., 1991; Pai, *et al*., 1990). Through guanosine

Molecular Machinery Regulating Exocytosis 65

from recycling endosomes to the plasma membrane, and restricts cargo trafficking, e.g. DE-

Rabs have important interactive functions at different stages of exocytosis. Protein sorting in recycling endosomes depends upon the function of the small GTPase Rab4, a close homologue of Rab11 (Li, *et al*., 2008; Ward, *et al*., 2005). Rab3 also has a role in anterograde traffic between the trans-Golgi network and recycling endosomes (Mohrmann, *et al*., 2002; van der Sluijs, *et al*., 1992). The Rab small GTPases, Sec4 in yeast and Rab11 in metazoans, facilitate trafficking of secretory vesicles carrying Sec15-exocyst components from recycling endosomes to the plasma membrane (Langevin, *et al*., 2005). Sec15 does not appear to interact with mammalian Rab4a, and therefore does not function as a Rab4 effector (Zhang, *et al*., 2004). This suggests a unique role for Rab11 in the final delivery of exocyst carrying secretory vesicles, to the plasma membrane (Chen, *et al*., 1998; Shandala, *et al*., 2011; Urbe, *et al*., 1993; Ward, *et al*., 2005). Sec15 does however interact with Rab3, a closely related homologue of Rab11, both of which appear to play a critical role in: secretory vesicle biogenesis; docking and priming of specialised secretory vesicles; delivery of synaptic and dense core vesicles to the active zone of exocytosis; and in maintaining a primed pool of vesicles available for rapid release (Schonn, *et al*., 2010; S. Wu, *et al*., 2005). Thus, the loss of Rab3 led to a reduction in the total number of synaptic vesicles as well as the number recruited to the active zone of the neural synapse (Gracheva, *et al*., 2008). Similarly, there was a reduction in the number of dense core vesicles docked at the plasma membrane in adrenal chromaffin cells, isolated from a mouse quadruple knockout lacking all four Rab3 A to D paralogues (Schonn, *et al*., 2010). An increased number of docked dense core vesicles was observed in PC12 and in adrenal chromaffin cells following Rab3 overexpression and this correlated with a strong inhibition of secretion (Holz, *et al*., 1994; Johannes, *et al*., 1994). Interestingly, there is evidence of some functional redundancy between Rab3 and its closest homologues, Rab27A and Rab27B, which are involved in the delivery of vesicles near the exocytic site (Fukuda, 2008; Gomi, *et al*., 2007; Ostrowski, *et al*., 2010). Studies of melanosome dynamics have indicated that Rab27A has a role in vesicular recruitment and this is mediated by its interaction with a specific effector called Melanophilin, which in turn binds an actin motor protein, MyosinVa (Hume & Seabra, 2011; Seabra & Coudrier, 2004). There appears to be a further functional divergence of Rabs, where Rab27A and Rab27B control different steps of the secretion pathway (Ostrowski, *et al*., 2010). Rab25, a close homologue of Rab11 with a different C-terminus, shows co-localization with Rab11 in exocytic/recycling vesicle membranes of some cells, and may function as a tissue specific

tethering factor (Calhoun & Goldenring, 1997; Khandelwal, *et al*., 2008).

molecular mechanism defining the plasma membrane docking sites?

Recent studies have implicated Rabs in the movement of transport vesicles from their site of formation to their site of fusion, and several Rabs have been linked to specific microtubuleor actin-based motor proteins (Hammer & Wu, 2002; Lapierre, *et al*., 2001) The role of Rabs in docking of secretory vesicles to the plasma membrane is mediated by their effectors (Fukuda, 2008). Thus, the small GTPases of the Rab family, through interplay with their specialist effector molecules, cooperatively target secretory vesicles from the trans-Golgi network (TGN) to the plasma membrane, and facilitate their docking at the active site of exocytosis (Fukuda, 2008; Orlando & Guo, 2009). This poses the question: what is the

Cadherin in *Drosophila* (Langevin, *et al*., 2005).

nucleotide-dependent conformational transitions within their G-protein domain (Pereira-Leal & Seabra, 2000), these GTPases act as molecular switches; cycling between the inactive GDP bound form and a GTP-bound active form, the process which regulates the activity of downstream effectors. This activity switch can be triggered by a variety of intracellular stimuli, most notably calcium ions (Khvotchev, *et al*., 2003; Zajac, *et al*., 2005). The current dogma suggests that guanosine-triphosphate-dependent activation is essential for Rho, Ral and Rab relocation to target membranes, which then triggers their function.

#### **3.1 Rab GTPases and vesicular tethering**

The Rab family of small GTPases is defined by the presence of at least one of five characteristic Rab motifs, with the RabF1 motif frequently positioned within the effector binding domain, and the RabF2 motif usually in the GTPase domain (Pereira-Leal & Seabra, 2000). Recent bioinformatic analysis of the Rab family using the "Rabifier" and "RabDB" tools have uncovered an interesting phenomenon, namely the highly dynamic evolution of this family, with a significant expansion and specialization of the Rabs involved in the secretory pathway (Diekmann, *et al*., 2011). The repertoire of secretion-related Rabs includes 14 subfamilies, which co-evolved with *Metazoan* multicellularity and may reflect either unique roles in tissue-specific membrane trafficking events or restricted trafficking of specialist vesicles (Diekmann, *et al*., 2011). The animal-specific subfamilies have purported roles in the regulation of secretion (e.g. Rab3, Rab26, Rab27, Rab33, Rab37, Rab39), while there are also Golgi-specific Rabs (Rab30, Rab33, Rab34, Rab43) and Rabs relating to the traffic to (Rab43) and from (Rab10) the Golgi. Rab proteins usually play positive roles in anterograde membrane trafficking, but the exact nature of their involvement (in vesicle budding, biogenesis, transport, docking, priming and fusion) depends on the particular pathway, and is yet to be defined. One of the most evolutionary conserved proteins, Rab11, appears to be associated with both constitutive and regulated secretory pathways, as shown in mammalian and insect cells (Chen, *et al*., 1998; Shandala, *et al*., 2011; Urbe, *et al*., 1993; Ward, *et al*., 2005).

There have been mechanistic links established between some Rab proteins and components of the exocyst complex. For example, the interaction of Sec15 with Rab proteins appears to be essential for the tethering of exocyst components to designated membranes. In yeast, the small Rab GTPase Sec4 (orthologous to mammalian Rab10) may bring Sec15 to the vesicular membrane (Guo, *et al*., 1999), which is an essential step in the tethering and assembly of the exocyst complex (Zajac, *et al*., 2005). Metazoan Sec15 is a known effector of Rab11 (S. Wu, *et al*., 2005; X. M. Zhang, *et al*., 2004). Through its C-terminal domain, *Drosophila* Sec15 can interact with Rab11 (and is found co-localized with Rab11 in *Drosophila* photoreceptor and sensory organ precursor cells (Jafar-Nejad, *et al*., 2005; S. Wu, *et al*., 2005)), as well as with Rab3, Rab8, and Rab27 (S. Wu, *et al*., 2005). The functional relationship between Sec15 and Rab11 also exists in mammalian cells (Langevin, *et al*., 2005; Zhang, *et al*., 2004), where the interaction with Rab11 is involved in sequestering the exocyst complex to the endosome recycling compartment. Interestingly, there is a functional co-dependence of Rab11 and Sec15, where the loss of Sec15 function affects the intracellular localisation of Rab11, and mimics a phenotype of abnormal Rab11 function. Evidence of this interaction can be observed during the dramatic changes that occur in photoreceptor cell development (S. Wu, *et al*., 2005). More specifically, the mutant *Sec15* phenotype involves impaired trafficking

nucleotide-dependent conformational transitions within their G-protein domain (Pereira-Leal & Seabra, 2000), these GTPases act as molecular switches; cycling between the inactive GDP bound form and a GTP-bound active form, the process which regulates the activity of downstream effectors. This activity switch can be triggered by a variety of intracellular stimuli, most notably calcium ions (Khvotchev, *et al*., 2003; Zajac, *et al*., 2005). The current dogma suggests that guanosine-triphosphate-dependent activation is essential for Rho, Ral

The Rab family of small GTPases is defined by the presence of at least one of five characteristic Rab motifs, with the RabF1 motif frequently positioned within the effector binding domain, and the RabF2 motif usually in the GTPase domain (Pereira-Leal & Seabra, 2000). Recent bioinformatic analysis of the Rab family using the "Rabifier" and "RabDB" tools have uncovered an interesting phenomenon, namely the highly dynamic evolution of this family, with a significant expansion and specialization of the Rabs involved in the secretory pathway (Diekmann, *et al*., 2011). The repertoire of secretion-related Rabs includes 14 subfamilies, which co-evolved with *Metazoan* multicellularity and may reflect either unique roles in tissue-specific membrane trafficking events or restricted trafficking of specialist vesicles (Diekmann, *et al*., 2011). The animal-specific subfamilies have purported roles in the regulation of secretion (e.g. Rab3, Rab26, Rab27, Rab33, Rab37, Rab39), while there are also Golgi-specific Rabs (Rab30, Rab33, Rab34, Rab43) and Rabs relating to the traffic to (Rab43) and from (Rab10) the Golgi. Rab proteins usually play positive roles in anterograde membrane trafficking, but the exact nature of their involvement (in vesicle budding, biogenesis, transport, docking, priming and fusion) depends on the particular pathway, and is yet to be defined. One of the most evolutionary conserved proteins, Rab11, appears to be associated with both constitutive and regulated secretory pathways, as shown in mammalian and insect cells (Chen, *et al*., 1998; Shandala, *et al*., 2011; Urbe, *et al*., 1993;

There have been mechanistic links established between some Rab proteins and components of the exocyst complex. For example, the interaction of Sec15 with Rab proteins appears to be essential for the tethering of exocyst components to designated membranes. In yeast, the small Rab GTPase Sec4 (orthologous to mammalian Rab10) may bring Sec15 to the vesicular membrane (Guo, *et al*., 1999), which is an essential step in the tethering and assembly of the exocyst complex (Zajac, *et al*., 2005). Metazoan Sec15 is a known effector of Rab11 (S. Wu, *et al*., 2005; X. M. Zhang, *et al*., 2004). Through its C-terminal domain, *Drosophila* Sec15 can interact with Rab11 (and is found co-localized with Rab11 in *Drosophila* photoreceptor and sensory organ precursor cells (Jafar-Nejad, *et al*., 2005; S. Wu, *et al*., 2005)), as well as with Rab3, Rab8, and Rab27 (S. Wu, *et al*., 2005). The functional relationship between Sec15 and Rab11 also exists in mammalian cells (Langevin, *et al*., 2005; Zhang, *et al*., 2004), where the interaction with Rab11 is involved in sequestering the exocyst complex to the endosome recycling compartment. Interestingly, there is a functional co-dependence of Rab11 and Sec15, where the loss of Sec15 function affects the intracellular localisation of Rab11, and mimics a phenotype of abnormal Rab11 function. Evidence of this interaction can be observed during the dramatic changes that occur in photoreceptor cell development (S. Wu, *et al*., 2005). More specifically, the mutant *Sec15* phenotype involves impaired trafficking

and Rab relocation to target membranes, which then triggers their function.

**3.1 Rab GTPases and vesicular tethering** 

Ward, *et al*., 2005).

from recycling endosomes to the plasma membrane, and restricts cargo trafficking, e.g. DE-Cadherin in *Drosophila* (Langevin, *et al*., 2005).

Rabs have important interactive functions at different stages of exocytosis. Protein sorting in recycling endosomes depends upon the function of the small GTPase Rab4, a close homologue of Rab11 (Li, *et al*., 2008; Ward, *et al*., 2005). Rab3 also has a role in anterograde traffic between the trans-Golgi network and recycling endosomes (Mohrmann, *et al*., 2002; van der Sluijs, *et al*., 1992). The Rab small GTPases, Sec4 in yeast and Rab11 in metazoans, facilitate trafficking of secretory vesicles carrying Sec15-exocyst components from recycling endosomes to the plasma membrane (Langevin, *et al*., 2005). Sec15 does not appear to interact with mammalian Rab4a, and therefore does not function as a Rab4 effector (Zhang, *et al*., 2004). This suggests a unique role for Rab11 in the final delivery of exocyst carrying secretory vesicles, to the plasma membrane (Chen, *et al*., 1998; Shandala, *et al*., 2011; Urbe, *et al*., 1993; Ward, *et al*., 2005). Sec15 does however interact with Rab3, a closely related homologue of Rab11, both of which appear to play a critical role in: secretory vesicle biogenesis; docking and priming of specialised secretory vesicles; delivery of synaptic and dense core vesicles to the active zone of exocytosis; and in maintaining a primed pool of vesicles available for rapid release (Schonn, *et al*., 2010; S. Wu, *et al*., 2005). Thus, the loss of Rab3 led to a reduction in the total number of synaptic vesicles as well as the number recruited to the active zone of the neural synapse (Gracheva, *et al*., 2008). Similarly, there was a reduction in the number of dense core vesicles docked at the plasma membrane in adrenal chromaffin cells, isolated from a mouse quadruple knockout lacking all four Rab3 A to D paralogues (Schonn, *et al*., 2010). An increased number of docked dense core vesicles was observed in PC12 and in adrenal chromaffin cells following Rab3 overexpression and this correlated with a strong inhibition of secretion (Holz, *et al*., 1994; Johannes, *et al*., 1994). Interestingly, there is evidence of some functional redundancy between Rab3 and its closest homologues, Rab27A and Rab27B, which are involved in the delivery of vesicles near the exocytic site (Fukuda, 2008; Gomi, *et al*., 2007; Ostrowski, *et al*., 2010). Studies of melanosome dynamics have indicated that Rab27A has a role in vesicular recruitment and this is mediated by its interaction with a specific effector called Melanophilin, which in turn binds an actin motor protein, MyosinVa (Hume & Seabra, 2011; Seabra & Coudrier, 2004). There appears to be a further functional divergence of Rabs, where Rab27A and Rab27B control different steps of the secretion pathway (Ostrowski, *et al*., 2010). Rab25, a close homologue of Rab11 with a different C-terminus, shows co-localization with Rab11 in exocytic/recycling vesicle membranes of some cells, and may function as a tissue specific tethering factor (Calhoun & Goldenring, 1997; Khandelwal, *et al*., 2008).

Recent studies have implicated Rabs in the movement of transport vesicles from their site of formation to their site of fusion, and several Rabs have been linked to specific microtubuleor actin-based motor proteins (Hammer & Wu, 2002; Lapierre, *et al*., 2001) The role of Rabs in docking of secretory vesicles to the plasma membrane is mediated by their effectors (Fukuda, 2008). Thus, the small GTPases of the Rab family, through interplay with their specialist effector molecules, cooperatively target secretory vesicles from the trans-Golgi network (TGN) to the plasma membrane, and facilitate their docking at the active site of exocytosis (Fukuda, 2008; Orlando & Guo, 2009). This poses the question: what is the molecular mechanism defining the plasma membrane docking sites?

Molecular Machinery Regulating Exocytosis 67

leads to the disassembly of the exocyst complex (Moskalenko, *et al*., 2003). Activation of the exocyst complex is initiated by the binding of Ral to Sec5 and Exo84 (Mott, *et al*., 2003). This is followed by the assembly of the full octameric exocyst complex at the interface of the vesicular and plasma membranes (Moskalenko, *et al*., 2003). Thus, the interaction of the exocyst complex with Ral is an essential step in anchoring secretory vesicles to the exocytosis-competent microdomains of the plasma membrane (Angus, *et al*., 2003; Fukai, *et* 

The functional interaction of Ral and the exocyst complex is highlighted by their coinvolvement in multiple exocytic processes. The exocyst complex has well-established roles in anterograde trafficking of membrane receptors from recycling endosomes (Langevin, *et al*., 2005; Xiong, *et al*., 2012; Yeaman, *et al*., 2004); membrane delivery in cell growth (Chernyshova, *et al*., 2011; Genre, *et al*., 2011); and the translocation of glucose transporters in response to insulin (Ljubicic, *et al*., 2009; Lopez, *et al*., 2008). Each of these processes has been linked to a functional requirement for a member of the Ral protein family. RalA is required for establishing neuronal cell polarity (Lalli, 2009), and the regulation of readily releasable pools of synaptic vesicles (Lee, *et al*., 2002; Li, *et al*., 2007). In the epithelium, RalB is required for delivery of membrane to the dynamic leading edge of migrating cells (Rosse, *et al*., 2006); while RalA is involved in polarised delivery of the membrane protein, E-Cadherin, to the basolateral surface of epithelial cells (Shipitsin & Feig, 2004). Exocytosis of vesicular content, such as hormones, chemokines, enzymes, and adhesion molecules from Weibel-Palade bodies (endothelial cell-specific storage organelles), occurs in response to a specific agonist that requires Ral regulation (Kim, *et al*., 2010; Rondaij, *et al*., 2008). RalA is required in glucose regulation where it mediates insulin secretion from pancreatic cells (Ljubicic, *et al*., 2009; Lopez, *et al*., 2008), and translocation of the glucose transporter GLUT4 in adipocytes (Chen, *et al*., 2007). Ral is also required for dense granule secretion from platelets (Kawato, *et al*., 2008) and cell growth and migration, all of which have been shown to be reliant on Ral for lipid raft trafficking to the plasma membrane (Balasubramanian, *et* 

Given that multiple GTPases regulate the assembly of the full octameric exocyst complex, which is necessary for vesicular tethering to the site of fusion, the assembly of the exocyst complex might represent the integration of various cellular signaling pathways that ensure

The exocyst mediated tethering of secretory vesicles to specific sites of the plasma membrane precedes the assembly of SNARE complexes and membrane fusion (He & Guo, 2009; Novick & Guo, 2002) (Figures 1 & 2). In the early 1980s, Rothman and colleagues used an *in vitro* trafficking assay to identify the soluble factors; *N*-ethylmaleimide-sensitive factor (NSF) and Soluble NSF Attachment Protein (SNAP) (Balch, *et al*., 1984). This was followed by isolation of their membrane receptors termed SNAREs (for SNAP receptors) (Sollner, *et al*., 1993). SNAREs were initially isolated from mammalian brain cells, as factors crucial for vesicle fusion–mediated release of neurotransmitters at synapses. It soon became evident that SNAREs are involved in most, if not all vesicular fusion events (Malsam, *et al*., 2008).

**4. Exocyst, SNARE complexes and membrane fusion machinery** 

*al*., 2003; Jin, *et al*., 2005; Mark, *et al*., 1996; Moskalenko, *et al*., 2002).

*al*., 2010; Spiczka & Yeaman, 2008).

tight control of exocytosis (Sugihara, *et al*., 2002).

#### **3.2 Rho GTPases and assembly of the plasma membrane exocyst complex**

The most highly conserved and best studied members of the Rho family, Rho1/A, Rac1 and Cdc42, play a crucial role in tethering and fusion of vesicles during regulated exocytosis (Ory & Gasman, 2011; Ridley, 2006; Williams, *et al*., 2009). Most Rho GTPases transiently localize at the plasma membrane, after being targeted to specific phosphoinositide-containing sub-domains. On the one hand, the Rho GTPases, Rho1/A and Rac1, are thought to regulate secretion by remodelling microtubules and the membrane-associated actin cytoskeleton (Ory & Gasman, 2011; Williams, *et al*., 2009). On the other hand, recent findings have implicated yeast Cdc42, Rho1/A and Rho3/C and mammalian TC10 in actin-independent regulation of exocytosis by anchoring the plasma membrane exocyst components, Sec3 and Exo70, to specific plasma membrane microdomains (Bendezu & Martin, 2011; Guo, *et al*., 2001; He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007; Moskalenko, *et al*., 2002; Novick & Guo, 2002; Wu & Brennwald, 2010; H. Wu, *et al*., 2010; Xiong, *et al*., 2012; Zajac, *et al*., 2005). However, in this case, the normal functioning of the Sec3 and Exo70 plasma membrane exocyst components is a prerequisite for the correct localization of cell polarity regulators such as Cdc42 (Zajac, *et al*., 2005). This might be due to the fact that Sec3 and Exo70 could independently bind to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), via their Cterminal D domain, thereby forming a plasma membrane targeting patch for exocytic proteins (He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007). Moreover, Exo70 was found to be directly associated with type Iγ phosphatidylinositol phosphate kinase (PIPKIγ), which facilitates the generation of a PI(4,5)P2 phospholipid microdomain and recruitment of Exo70 to the plasma membrane (Xiong, *et al*., 2012). Thus, the cooperation between the Sec3 and Exo70 exocyst components and Rho small GTPases defines competent sites for exocytosis. The next question is: how are secretory vesicles targeted to these sites?

#### **3.3 Ral small GTPases and the exocyst complex**

Ras-like (Ral) small GTPase was first discovered in human platelet cells in 1991 (van der Meulen, *et al*., 1991), where its association with dense granules suggested a potential regulatory role in the release of storage contents from these granules (Mark, *et al*., 1996). Subsequently, Ral small GTPases were linked to exocytosis in neural, epithelial, endothelial endocrinal, and pancreatic tissues (Hazelett, *et al*., 2011; Lopez, *et al*., 2008; Moskalenko, *et al*., 2003; Polzin, *et al*., 2002; Rondaij, *et al*., 2004; Rondaij, *et al*., 2008; Takaya, *et al*., 2007). The two mammalian Ral homologues RalA and RalB share 85% protein sequence identity, and are well conserved in evolution (van Dam & Robinson, 2006). The bulk of the Ral protein comprises a conserved nucleotide phosphate-binding motif (Marchler-Bauer, *et al*., 2011; van Dam & Robinson, 2006). RalA, but not RalB, contains a short amphipathic helix that binds the Ca2+-sensing protein Calmodulin, conferring Ca2+-dependent activation of RalA during regulated exocytosis (van Dam & Robinson, 2006). Ral functions as an essential component of the cellular machinery, regulating the post-Golgi processing of secretory vesicle membrane, via activation of the exocyst complex (X. W. Chen, *et al*., 2011a; Feig, 2003; Kawato, *et al*., 2008; Mark, *et al*., 1996; Mott, *et al*., 2003). It has been suggested that GTPbound Ral, through its effectors Sec5 and Exo84, brings together the plasma and vesicular membrane exocyst subunits, as the loss of RalA, or mutation of its effector binding motif,

The most highly conserved and best studied members of the Rho family, Rho1/A, Rac1 and Cdc42, play a crucial role in tethering and fusion of vesicles during regulated exocytosis (Ory & Gasman, 2011; Ridley, 2006; Williams, *et al*., 2009). Most Rho GTPases transiently localize at the plasma membrane, after being targeted to specific phosphoinositide-containing sub-domains. On the one hand, the Rho GTPases, Rho1/A and Rac1, are thought to regulate secretion by remodelling microtubules and the membrane-associated actin cytoskeleton (Ory & Gasman, 2011; Williams, *et al*., 2009). On the other hand, recent findings have implicated yeast Cdc42, Rho1/A and Rho3/C and mammalian TC10 in actin-independent regulation of exocytosis by anchoring the plasma membrane exocyst components, Sec3 and Exo70, to specific plasma membrane microdomains (Bendezu & Martin, 2011; Guo, *et al*., 2001; He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007; Moskalenko, *et al*., 2002; Novick & Guo, 2002; Wu & Brennwald, 2010; H. Wu, *et al*., 2010; Xiong, *et al*., 2012; Zajac, *et al*., 2005). However, in this case, the normal functioning of the Sec3 and Exo70 plasma membrane exocyst components is a prerequisite for the correct localization of cell polarity regulators such as Cdc42 (Zajac, *et al*., 2005). This might be due to the fact that Sec3 and Exo70 could independently bind to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), via their Cterminal D domain, thereby forming a plasma membrane targeting patch for exocytic proteins (He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007). Moreover, Exo70 was found to be directly associated with type Iγ phosphatidylinositol phosphate kinase (PIPKIγ), which facilitates the generation of a PI(4,5)P2 phospholipid microdomain and recruitment of Exo70 to the plasma membrane (Xiong, *et al*., 2012). Thus, the cooperation between the Sec3 and Exo70 exocyst components and Rho small GTPases defines competent sites for exocytosis. The next question is: how are secretory vesicles

Ras-like (Ral) small GTPase was first discovered in human platelet cells in 1991 (van der Meulen, *et al*., 1991), where its association with dense granules suggested a potential regulatory role in the release of storage contents from these granules (Mark, *et al*., 1996). Subsequently, Ral small GTPases were linked to exocytosis in neural, epithelial, endothelial endocrinal, and pancreatic tissues (Hazelett, *et al*., 2011; Lopez, *et al*., 2008; Moskalenko, *et al*., 2003; Polzin, *et al*., 2002; Rondaij, *et al*., 2004; Rondaij, *et al*., 2008; Takaya, *et al*., 2007). The two mammalian Ral homologues RalA and RalB share 85% protein sequence identity, and are well conserved in evolution (van Dam & Robinson, 2006). The bulk of the Ral protein comprises a conserved nucleotide phosphate-binding motif (Marchler-Bauer, *et al*., 2011; van Dam & Robinson, 2006). RalA, but not RalB, contains a short amphipathic helix that binds the Ca2+-sensing protein Calmodulin, conferring Ca2+-dependent activation of RalA during regulated exocytosis (van Dam & Robinson, 2006). Ral functions as an essential component of the cellular machinery, regulating the post-Golgi processing of secretory vesicle membrane, via activation of the exocyst complex (X. W. Chen, *et al*., 2011a; Feig, 2003; Kawato, *et al*., 2008; Mark, *et al*., 1996; Mott, *et al*., 2003). It has been suggested that GTPbound Ral, through its effectors Sec5 and Exo84, brings together the plasma and vesicular membrane exocyst subunits, as the loss of RalA, or mutation of its effector binding motif,

**3.2 Rho GTPases and assembly of the plasma membrane exocyst complex** 

targeted to these sites?

**3.3 Ral small GTPases and the exocyst complex** 

leads to the disassembly of the exocyst complex (Moskalenko, *et al*., 2003). Activation of the exocyst complex is initiated by the binding of Ral to Sec5 and Exo84 (Mott, *et al*., 2003). This is followed by the assembly of the full octameric exocyst complex at the interface of the vesicular and plasma membranes (Moskalenko, *et al*., 2003). Thus, the interaction of the exocyst complex with Ral is an essential step in anchoring secretory vesicles to the exocytosis-competent microdomains of the plasma membrane (Angus, *et al*., 2003; Fukai, *et al*., 2003; Jin, *et al*., 2005; Mark, *et al*., 1996; Moskalenko, *et al*., 2002).

The functional interaction of Ral and the exocyst complex is highlighted by their coinvolvement in multiple exocytic processes. The exocyst complex has well-established roles in anterograde trafficking of membrane receptors from recycling endosomes (Langevin, *et al*., 2005; Xiong, *et al*., 2012; Yeaman, *et al*., 2004); membrane delivery in cell growth (Chernyshova, *et al*., 2011; Genre, *et al*., 2011); and the translocation of glucose transporters in response to insulin (Ljubicic, *et al*., 2009; Lopez, *et al*., 2008). Each of these processes has been linked to a functional requirement for a member of the Ral protein family. RalA is required for establishing neuronal cell polarity (Lalli, 2009), and the regulation of readily releasable pools of synaptic vesicles (Lee, *et al*., 2002; Li, *et al*., 2007). In the epithelium, RalB is required for delivery of membrane to the dynamic leading edge of migrating cells (Rosse, *et al*., 2006); while RalA is involved in polarised delivery of the membrane protein, E-Cadherin, to the basolateral surface of epithelial cells (Shipitsin & Feig, 2004). Exocytosis of vesicular content, such as hormones, chemokines, enzymes, and adhesion molecules from Weibel-Palade bodies (endothelial cell-specific storage organelles), occurs in response to a specific agonist that requires Ral regulation (Kim, *et al*., 2010; Rondaij, *et al*., 2008). RalA is required in glucose regulation where it mediates insulin secretion from pancreatic cells (Ljubicic, *et al*., 2009; Lopez, *et al*., 2008), and translocation of the glucose transporter GLUT4 in adipocytes (Chen, *et al*., 2007). Ral is also required for dense granule secretion from platelets (Kawato, *et al*., 2008) and cell growth and migration, all of which have been shown to be reliant on Ral for lipid raft trafficking to the plasma membrane (Balasubramanian, *et al*., 2010; Spiczka & Yeaman, 2008).

Given that multiple GTPases regulate the assembly of the full octameric exocyst complex, which is necessary for vesicular tethering to the site of fusion, the assembly of the exocyst complex might represent the integration of various cellular signaling pathways that ensure tight control of exocytosis (Sugihara, *et al*., 2002).
