**Molecular Machinery Regulating Exocytosis**

T. Shandala, R. Kakavanos-Plew, Y.S. Ng, C. Bader, A. Sorvina, E.J. Parkinson-Lawrence, R.D. Brooks, G.N. Borlace, M.J. Prodoehl and D.A. Brooks *Mechanisms in Cell Biology and Diseases Research Group, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia Australia* 

#### **1. Introduction**

60 Crosstalk and Integration of Membrane Trafficking Pathways

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Exocytosis is the major intracellular route for the delivery of proteins and lipids to the plasma membrane and the means by which vesicular contents are released into the extracellular space. The anterograde trafficking of vesicles to the plasma membrane is vital for membrane expansion during cell division; cell growth and migration; the delivery of specialised molecules to establish cell polarity; cell-to-cell communication; neurotransmission and the secretion of response factors such as hormones, cytokines and antimicrobial peptides. There are two major trafficking routes in eukaryotic cells, which are referred to as constitutive and regulated (Ory & Gasman, 2011). Constitutive exocytosis involves the steady state delivery of secretory carrier vesicles from the endoplasmic reticulum via the Golgi apparatus to the plasma membrane (Lacy & Stow, 2011). Regulated or granule-mediated exocytosis involves a specific trigger, usually a burst of intracellular calcium following an extrinsic stimulus. This system is utilized for secretion in neuronal cells and other specialist secretory cells, such as neuroendocrine, endocrine and exocrine cells (Burgoyne & Morgan, 2003; Jolly & Sattentau, 2007; Lacy & Stow, 2011). Regulated exocytosis enables a rapid response from a subpopulation of vesicles already primed and competent for fusion (Manjithaya & Subramani, 2011; Nickel & Seedorf, 2008; Nickel, 2010). Regulated exocytosis is also used for polarised traffic of vesicular membrane and cargo to specific spatial landmarks and this is particularly important during times of dramatic change in cell morphology, such as cell division, cell motility, phagocytosis and axonal outgrowth.

Regulated exocytosis involves the shuttling of carrier vesicles between vesicular compartments, as they are transported towards the plasma membrane. Each step in this process requires the fission of a vesicle from a donor compartment. This carrier vesicle is then targeted/trafficked to an acceptor compartment where docking and fusion takes place, and the cargo is either unloaded or further processed (Bonifacino & Glick, 2004). These fission and fusion steps are repeated until the cargo reaches the plasma membrane (Bonifacino & Glick, 2004). This sequential trafficking of secretory vesicles is orchestrated by a complex set of molecular machinery including: small GTPases of the Ral, Rab and Rho subfamilies that regulate the processes of vesicle formation, traffic and fusion; the

Molecular Machinery Regulating Exocytosis 63

Fig. 1. Post-translational regulation of exocytic vesicle tethering via the exocyst complex

Zhang, *et al*., 2005).

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

mechanism for its plasma membrane localization (He, *et al*., 2007; Moskalenko, *et al*., 2002; H. Wu, *et al*., 2010), which is distinct from the Rab-dependent targeting of Sec15 to the vesicular membrane (Guo, *et al*., 1999; Langevin, *et al*., 2005; S. Wu, *et al*., 2005; Zhang, *et al*., 2004). Co-assembly of these two exocyst sub-complexes to form the entire complex is governed by Ral-GTPase via its interaction with Sec5 (Hohlfeld, 1990). Prior to membrane fusion, SNAREs (e.g. Sec1, Sro7p and Sro77p) interact with the exocyst complex (via Sec6, Exo84) to facilitate fusion between the vesicle and plasma membranes (Morgera, *et al*., 2012;

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

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

requirements for small GTPase subfamily members during exocytosis (Figure 1).

exocyst complex for vesicle assembly and membrane tethering; and soluble *N*ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins for vesicular fusion. At the target domain of the plasma membrane, cross-talk between the exocyst complex and SNARE proteins culminates in vesicle-to-plasma membrane fusion, and thereby delivery of membrane proteins and luminal cargo. There are many posttranslational modifications of the vesicular machinery that facilitate exocytosis. These include the addition of lipid moieties to increase membrane binding affinity, the switching of GTPase activity by nucleotide exchange factors, phosphorylation, and ubiquitination. Phosphorylation is of particular importance as it incorporates the vesicular trafficking machinery into a circuit of cellular signaling cascades. This chapter focuses on the process of exocytosis and the regulatory role that post-translational modification has on the exocytic machinery. Because the small GTPases and the exocyst complex have multiple inter-connected functions during vesicle formation, trafficking and fusion, we have focused discussion here to the final steps of the exocytic process, which occur in close proximity to the plasma membrane.

## **2. The exocyst complex and vesicle interaction with the plasma membrane**

The exocyst is a scaffolding complex that is required for the final steps of regulated, and constitutive exocytosis (Hsu, *et al*., 2004). The exocyst complex is attached to the cytosolic face of the exocytic vesicular membrane, and tethers the vesicle to specific domains of the plasma membrane (Brymora, *et al*., 2001; X. W. Chen, *et al*., 2011a; Fukai, *et al*., 2003; Inoue, *et al*., 2003; Li, *et al*., 2007; Moskalenko, *et al*., 2002) (Figure 1). The pioneering studies of the early 1990's discovered that there are six yeast secretion (Sec) proteins; Sec3, Sec5, Sec6, Sec8, Sec10 and Sec15; and two exocyst (Exo) subunit proteins; Exo70 and Exo84, which form the exocyst complex (TerBush, *et al*., 1996). The constituents of the exocyst complex are conserved between yeast and mammals (He & Guo, 2009) and there are striking structural and topological similarities in the C-terminal domains of Sec6, Sec15, Exo70 and Exo84, despite there being less than 10% sequence identity between the individual proteins. These C-terminal domains consist of multiple rod-like helical bundles, which appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins (Sivaram, *et al*., 2006). The interaction between these helical structures may create the framework that is necessary for the assembly of the exocyst complex (Munson & Novick, 2006).

There is some evidence that the exocyst complex may be present as distinct sub-complexes on vesicular and plasma membranes. In yeast, two members of the complex are associated with the plasma membrane; Sec3 and Exo70, while in mammals only Exo70 appears to be found on the plasma membrane (He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007). It is likely that the membrane localisation of Sec3 and Exo70 controls targeting of secretory vesicles to distinct domains of the plasma membrane, thereby defining the sites of active exocytosis and membrane growth during cell migration and cytokinesis (Liu & Guo, 2011). It has been suggested that the Sec3 and Exo70 plasma membrane complex also contains Sec5, Sec8 and Sec6, while Exo84, Sec10 and Sec15 are complexed to the vesicle membrane (Moskalenko, *et al*., 2003). By binding to the vesicular membrane, Sec15 initiates the assembly of the vesicular exocyst sub-complex, while Sec3 and Exo70 mediate assembly of the plasma membrane sub-complex. Sec3 relies on a Rho-mediated targeting

exocyst complex for vesicle assembly and membrane tethering; and soluble *N*ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins for vesicular fusion. At the target domain of the plasma membrane, cross-talk between the exocyst complex and SNARE proteins culminates in vesicle-to-plasma membrane fusion, and thereby delivery of membrane proteins and luminal cargo. There are many posttranslational modifications of the vesicular machinery that facilitate exocytosis. These include the addition of lipid moieties to increase membrane binding affinity, the switching of GTPase activity by nucleotide exchange factors, phosphorylation, and ubiquitination. Phosphorylation is of particular importance as it incorporates the vesicular trafficking machinery into a circuit of cellular signaling cascades. This chapter focuses on the process of exocytosis and the regulatory role that post-translational modification has on the exocytic machinery. Because the small GTPases and the exocyst complex have multiple inter-connected functions during vesicle formation, trafficking and fusion, we have focused discussion here to the final steps of the exocytic process, which occur in

**2. The exocyst complex and vesicle interaction with the plasma membrane**  The exocyst is a scaffolding complex that is required for the final steps of regulated, and constitutive exocytosis (Hsu, *et al*., 2004). The exocyst complex is attached to the cytosolic face of the exocytic vesicular membrane, and tethers the vesicle to specific domains of the plasma membrane (Brymora, *et al*., 2001; X. W. Chen, *et al*., 2011a; Fukai, *et al*., 2003; Inoue, *et al*., 2003; Li, *et al*., 2007; Moskalenko, *et al*., 2002) (Figure 1). The pioneering studies of the early 1990's discovered that there are six yeast secretion (Sec) proteins; Sec3, Sec5, Sec6, Sec8, Sec10 and Sec15; and two exocyst (Exo) subunit proteins; Exo70 and Exo84, which form the exocyst complex (TerBush, *et al*., 1996). The constituents of the exocyst complex are conserved between yeast and mammals (He & Guo, 2009) and there are striking structural and topological similarities in the C-terminal domains of Sec6, Sec15, Exo70 and Exo84, despite there being less than 10% sequence identity between the individual proteins. These C-terminal domains consist of multiple rod-like helical bundles, which appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins (Sivaram, *et al*., 2006). The interaction between these helical structures may create the framework that is necessary for the assembly of the exocyst complex (Munson &

There is some evidence that the exocyst complex may be present as distinct sub-complexes on vesicular and plasma membranes. In yeast, two members of the complex are associated with the plasma membrane; Sec3 and Exo70, while in mammals only Exo70 appears to be found on the plasma membrane (He, *et al*., 2007; He & Guo, 2009; Inoue, *et al*., 2003; J. Liu, *et al*., 2007). It is likely that the membrane localisation of Sec3 and Exo70 controls targeting of secretory vesicles to distinct domains of the plasma membrane, thereby defining the sites of active exocytosis and membrane growth during cell migration and cytokinesis (Liu & Guo, 2011). It has been suggested that the Sec3 and Exo70 plasma membrane complex also contains Sec5, Sec8 and Sec6, while Exo84, Sec10 and Sec15 are complexed to the vesicle membrane (Moskalenko, *et al*., 2003). By binding to the vesicular membrane, Sec15 initiates the assembly of the vesicular exocyst sub-complex, while Sec3 and Exo70 mediate assembly of the plasma membrane sub-complex. Sec3 relies on a Rho-mediated targeting

close proximity to the plasma membrane.

Novick, 2006).

Fig. 1. Post-translational regulation of exocytic vesicle tethering via the exocyst complex

mechanism for its plasma membrane localization (He, *et al*., 2007; Moskalenko, *et al*., 2002; H. Wu, *et al*., 2010), which is distinct from the Rab-dependent targeting of Sec15 to the vesicular membrane (Guo, *et al*., 1999; Langevin, *et al*., 2005; S. Wu, *et al*., 2005; Zhang, *et al*., 2004). Co-assembly of these two exocyst sub-complexes to form the entire complex is governed by Ral-GTPase via its interaction with Sec5 (Hohlfeld, 1990). Prior to membrane fusion, SNAREs (e.g. Sec1, Sro7p and Sro77p) interact with the exocyst complex (via Sec6, Exo84) to facilitate fusion between the vesicle and plasma membranes (Morgera, *et al*., 2012; Zhang, *et al*., 2005).
