**2. Exocytosis**

The exocytic pathway delivers cargo carrying vesicles from either the *trans-*Golgi network (TGN) or recycling endosomes to the plasma membrane, where membrane fusion occurs to release the vesicular content (Figure 1). This vesicular content may be either vesicle membrane proteins directed to the cell surface, or lumenal contents for secretion into the extracellular milieu. This anterograde trafficking route may vary depending upon the cargo and cell type involved (Wurster *et al*., 1990), such as in melanocytes for melanin exocytosis and in neurons for neurotransmission. Exocytosis is also involved in numerous other cellular functions, including immune responses, cell-cell communication, cell growth, cell polarity and neurotransmission.

There are two main exocytic routes from the *trans*-Golgi network to the plasma membrane: the constitutive and the regulated routes (Stow *et al*., 2009). The constitutive route continuously delivers membrane and cargo from the *trans*-Golgi network to the cell surface, and is thought to be utilized for housekeeping functions. Although this process can be upregulated in response to environmental stress, it is generally representative of a basal level of cell activity and secretion. However, a number of pro-inflammatory cytokines, including TNF and IL-6, are released from macrophages via the constitutive route, in response to pro-inflammatory stimuli (Shurety *et al*., 2000). The regulated route involves the redirection of newly synthesized cargo to compartments where these molecules are stored until their release is triggered by a specific stimulus (mediated by calcium ion mobilization). In this

cell surface (Franken *et al*., 2003). Finally, during the removal of dysfunctional exocytic vesicles or the rapid cessation of secretion (e.g. during neurotransmission), the cell requires organelle-specific molecular machinery, for the nucleation of autophagy (Geng *et al*., 2010). Despite defects in multiple human syndromes that demonstrate changes in both exocytosis and autophagy, the mutual dependence of these processes on common molecular machinery has only recently been investigated. Evidence indicates that the exocyst complex and its regulator Ral (Ras like GTPase), both of which are known to have a critical function in exocytosis, also appear to be essential for the initiation of autophagy (Bodemann *et al*., 2011). Similarly, the small GTPase Rab11 has a critical role in exocytosis at the recycling endosome and in exocytic vesicle function (van Ijzendoorn, 2006), although in times of starvation where autophagy is induced, Rab11 containing vesicular compartments have been shown to asscociate with autophagosomes (Rab11 positive amphisomes; (Fader *et al*., 2008)). Finally, disruption of the exocytic Rab GTPase Sec4 and its guanine nucleotide exchange factor Sec2, can have significant effects on the anterograde movement of the integral autophagosome membrane protein Atg9 (Autophagy related protein 9), thereby influencing the recruitment of Atg8 to the phagophore assembly site (PAS) (Bodemann *et al*., 2011). The movements of Atg9 and Atg8 are of particular interest as they are both important during the initiation of autophagy (Geng *et al*., 2010; Wang *et al*., 2009). Atg9 has, in turn, been reported to reside on exocytic vesicles that can be converted into a phagophore assembly site (Mari *et al*., 2010; Mari & Reggiori, 2010). The aim of this chapter is to provide an overview of the exocytic and autophagic processes with a focus on the common molecular machinery acting at critical control points. It is this machinery that may facilitate communication between these functionally distinct vesicular compartments

The exocytic pathway delivers cargo carrying vesicles from either the *trans-*Golgi network (TGN) or recycling endosomes to the plasma membrane, where membrane fusion occurs to release the vesicular content (Figure 1). This vesicular content may be either vesicle membrane proteins directed to the cell surface, or lumenal contents for secretion into the extracellular milieu. This anterograde trafficking route may vary depending upon the cargo and cell type involved (Wurster *et al*., 1990), such as in melanocytes for melanin exocytosis and in neurons for neurotransmission. Exocytosis is also involved in numerous other cellular functions, including immune responses, cell-cell communication, cell growth, cell

There are two main exocytic routes from the *trans*-Golgi network to the plasma membrane: the constitutive and the regulated routes (Stow *et al*., 2009). The constitutive route continuously delivers membrane and cargo from the *trans*-Golgi network to the cell surface, and is thought to be utilized for housekeeping functions. Although this process can be upregulated in response to environmental stress, it is generally representative of a basal level of cell activity and secretion. However, a number of pro-inflammatory cytokines, including TNF and IL-6, are released from macrophages via the constitutive route, in response to pro-inflammatory stimuli (Shurety *et al*., 2000). The regulated route involves the redirection of newly synthesized cargo to compartments where these molecules are stored until their release is triggered by a specific stimulus (mediated by calcium ion mobilization). In this

and may act as potential sites for regulation.

polarity and neurotransmission.

**2. Exocytosis** 

Fig. 1. Proposed model for crosstalk between autophagy and exocytosis.

route, cargo can be trafficked via a number of compartments including recycling endosomes, early endosomes, multivesicular bodies, secretory granules and secretory lysosomes. This pathway is utilized for the immune related secretion of cytokines and antimicrobial peptides, following exposure to pathogens or inflammatory stimuli. Similarly, for neurotransmitter release, exocytosis is stimulated by an increase in the intracellular calcium ion concentration in neurons; which allows the propagation of neuronal function. These different pathways are in dynamic balance with the endocytic pathway, which, apart from facilitating uptake into the cell, allows the recovery of membrane from the plasma membrane following exocytosis, enabling the cell to control its surface area (Khandelwal *et al*., 2010). The molecular machinery that drives exocytosis therefore operates in conjunction with the endocytic machinery, and in some cases may involve common elements that have dual function.

#### **2.1 The molecular machinery for exocytosis**

The molecular machinery that facilitates the process of exocytosis can vary with respect to the specific cell type and specialist cargo being transported, although two key molecular complexes are conserved for most membrane associated exocytic events; the exocyst and the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex (Liu & Guo, 2011; Nair *et al*., 2011). Through interaction with a number of effector molecules, these complexes mediate the tethering, docking and fusion of vesicles with the plasma membrane.

The exocyst is an octomeric complex that is required for the efficient delivery of exocytic vesicles to the plasma membrane (TerBush *et al*., 1996). The components of the exocyst complex were first identified for yeast in the early 1990's, with mammalian orthologues

At the Intersection of the Pathways for Exocytosis and Autophagy 113

provided evidence of interactions between SNARE proteins and components of the exocyst complex (Bao *et al*., 2008; Hattendorf *et al*., 2007; Wiederkehr *et al*., 2004; Zhang *et al*., 2005). In yeast, this interaction is orchestrated though WD-40 adaptor proteins Sro7p and Sro77p (Zhang *et al*., 2005), which are homologues of lethal giant larvae (Lgl); first identified as a tumor suppressor in *Drosophila* (Gateff, 1978) and since demonstrated to play a role in cell polarity (Bilder *et al*., 2000). Sro7p and Sro77p interact with the exocyst components Sec6 and Exo84 as well as t-SNARE Sec9, thus providing a link between these two complexes to

Autophagy is responsible for a number of routine housekeeping functions, including the elimination of defective proteins, the prevention of abnormal protein aggregate accumulation, the turnover of glycogen, the removal of intracellular pathogens and the recycling of aged or dysfunctional organelles. These functions are likely to be critical for autophagy-mediated protection against aging, cancer, neurodegenerative disease and infection (Levine & Kroemer, 2008). Autophagy involves the engulfing of cytoplasmic content into a double membrane vesicle, which is used to mediate the degradation of the internalised contents following interaction with endosome and lysosome compartments (Figure 1). Autophagy normally occurs at a basal level, but stimuli such as starvation, hormonal and developmental signals, accumulation of unfolded proteins or invasion of microorganisms, can each modulate the rate

The process of autophagy is mediated by the recruitment of autophagy related proteins to the limiting membranes of the forming phagosome, where they assemble the so-called preautophagosomal structure. This nucleation step is known to occur at sites adjacent to mitochondria in yeast (Mari & Reggiori, 2010), while other eukaryotes are thought to have multiple nucleation sites that may include the endoplasmic reticulum, Golgi, mitochondria and secretory vesicles (Hailey *et al*., 2010; Hamasaki & Yoshimori, 2010; Militello & Colombo, 2011; Tooze & Yoshimori, 2010; Weidberg *et al*., 2011). Autophagosomes are formed via the expansion of the isolation membrane to completely surround an area of cytoplasm. Maturation of the autophagosome involves fusion with a multivesicular body to form an amphisome, which subsequently fuses with a lysosome to become a fully functional autolysosome. Through the action of lysosomal enzymes, the degradation process then

**3.2 The molecular machinery involved in autophagosome formation and maturation**  The induction and nucleation of autophagy is dependent on the successive assembly of a number of complexes within the cytoplasm, such as the Atg1-Atg13 (or mammalian unc-51 like kinase 1(Ulk1)–Atg13) kinase complex and the Atg5-Atg12 ubiquitin-like conjugation system. Up-stream signalling pathways lead to the activation of the Atg1/Ulk1 complex, which in turn recruits other members of the autophagic machinery to the site of nucleation. The exact mode for this recruitment is as yet unknown, however the individual step-specific

mediate the final steps of membrane fusion and exocytosis (Zhang *et al*., 2005).

**3. Autophagy** 

of autophagic activity (Meijer & Codogno, 2004).

**3.1 The induction and sequence of the autophagic process** 

recycles molecular constituents back into the cytoplasm.

complexes are well described for yeast and higher eukaryote systems.

subsequently being identified (Lipschutz & Mostov, 2002). In yeast, this complex consists of six secretion related proteins (Sec), Sec3, Sec5, Sec6, Sec8, Sec10 and Sec15, with an additional two subunits known as exocyst related proteins (Exo), Exo70 and Exo84. Tethering to the plasma membrane is mediated by GTPase proteins, such as the yeast proteins, Rho3 and Cdc42 (He *et al*., 2007; He & Guo, 2009), or by TC10 in mammalian cells (Dupraz *et al*., 2009; Inoue *et al*., 2006; Pommereit & Wouters, 2007). Recognition of the exocytic vesicle by the exocyst is mediated by the Rab GTPase proteins, Sec4 in yeast (Guo *et al*., 1999; Zajac *et al*., 2005) or Rab11 in metazoans (Novick & Guo, 2002; Novick *et al*., 2006). In mammalian cells, assembly of this complex is controlled by RalA and RalB (Chen *et al*., 2011a; Chen *et al*., 2007; Chen *et al*., 2011b).

Assembly of the exocyst serves to tether exocytic vesicles to a specific plasma membrane site, demarcated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), Sec3 and Exo70 (He *et al*., 2007; He & Guo, 2009). The Sec3 and Exo70 membrane associated components of the exocyst act to target vesicles to the site of exocytosis, via the direct association of positively charged residues in the D domain at the C-terminus with PI(4,5)P2, in the plasma membrane (He *et al*., 2007; He & Guo, 2009). Multiple GTPases then regulate the assembly interface of a full octameric exocyst complex. The GTPase proteins Rho3 and Cdc42 also interact with Exo70 and Sec3 to facilitate the assembly of the exocyst complex at the plasma membrane (He *et al*., 2007; Moskalenko *et al*., 2002). In mammalian cells, Exo70 facilitates exocyst association with the plasma membrane through its interaction with TC10 (the orthologue of Cdc42) (Inoue *et al*., 2003; Liu *et al*., 2007). In yeast, secretory vesicles acquire the Rab GTPase protein Sec4, which directly interacts with the exocyst, via the Sec15 subunit, thus allowing the secretory vesicle to be recognised by the exocyst plasma membrane complex. Unlike in yeast, the vesicular targeting of the exocyst in metazoans is thought to occur through interactions between Sec15 and Rab11 (Langevin *et al*., 2005; Wu *et al*., 2005; Zhang *et al*., 2004), and the tethering of the secretory vesicles to the plasma membrane is regulated by Sec5 and Ral (Brymora *et al*., 2001; Chen *et al*., 2011a; Li *et al*., 2007). Active RalA (GTP bound form) interacts with Sec5, and upon delivery of the vesicles to the plasma membrane, the interaction between RalA-Sec5 is broken through the phosphorylation of Sec5 by protein kinase C (PKC) (Chen *et al*., 2011a). Detachment of Sec5 from RalA allows the release of the exocyst complex once the vesicle is delivered to the plasma membrane. The emerging model for the assembly of the exocyst suggests that the components are present as distinct sub-complexes on vesicles and the plasma membrane. In this manner, the assembly of the exocyst may integrate various cellular signalling pathways to ensure that exocytosis is tightly controlled (Sugihara *et al*., 2002).

Following cell surface membrane tethering by the exocyst complex, SNARE proteins facilitate the final step of exocytosis by bringing together the vesicular and plasma membranes for fusion. There are two groups of SNARE proteins; t-SNAREs, such as syntaxin1 and Sec9, which are found on the inner leaflet of the plasma membrane of cells and denote the target membrane; and v-SNAREs, which are found on a range of membrane compartments and denotes the vesicular membrane (Shorer *et al*., 2005; Stow *et al*., 2006). These proteins work by cognate pairing of t-SNAREs with their opposing v-SNAREs to form a four helix bundle, which allows the two membranes to be brought into close proximity, and this facilitates membrane fusion (Stow *et al*., 2006). A number of studies have provided evidence of interactions between SNARE proteins and components of the exocyst complex (Bao *et al*., 2008; Hattendorf *et al*., 2007; Wiederkehr *et al*., 2004; Zhang *et al*., 2005). In yeast, this interaction is orchestrated though WD-40 adaptor proteins Sro7p and Sro77p (Zhang *et al*., 2005), which are homologues of lethal giant larvae (Lgl); first identified as a tumor suppressor in *Drosophila* (Gateff, 1978) and since demonstrated to play a role in cell polarity (Bilder *et al*., 2000). Sro7p and Sro77p interact with the exocyst components Sec6 and Exo84 as well as t-SNARE Sec9, thus providing a link between these two complexes to mediate the final steps of membrane fusion and exocytosis (Zhang *et al*., 2005).
