**3. Autophagy**

112 Crosstalk and Integration of Membrane Trafficking Pathways

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*.,

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

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

2011a; Chen *et al*., 2007; Chen *et al*., 2011b).

(Sugihara *et al*., 2002).

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 of autophagic activity (Meijer & Codogno, 2004).

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

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 recycles molecular constituents back into the cytoplasm.

#### **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 complexes are well described for yeast and higher eukaryote systems.

At the Intersection of the Pathways for Exocytosis and Autophagy 115

Orange (Dor)/Vps18 and Carnation (Car)/Vps33a (Fader *et al*., 2008; Gutierrez *et al*., 2004a; Gutierrez *et al*., 2004b; Simonsen & Tooze, 2009), as well as the lysosomal membrane proteins, Lamp-1 and Lamp-2 (Tanaka *et al*., 2000). After fusion with the lysosome, degradation of protein, lipid, glycogen, RNA, DNA and other contents is dependent upon the action of lysosomal acid hydrolases (Koike *et al*., 2005; Tanaka *et al*., 2000; Tanida *et al*., 2005). The small molecules (e.g. amino acids and sugars) are then transported back to the cytosol for protein

Altered regulation of exocytosis and autophagy has been shown in a number of debilitating diseases including cancer (Gozuacik & Kimchi, 2006; Levine, 2007; Miracco *et al*., 2007; Pattingre *et al*., 2005; Tayeb *et al*., 2005), neurodegenerative diseases (Gao & Hong, 2008; Keating, 2008; Yu *et al*., 2005), and chronic inflammatory diseases (Barbier, 2003; Barrett, 2008;

In cancer, the uncontrolled cell proliferation that results in tumor outgrowth is associated with increased secretion of pro-oncogenic proteins and lysosomal enzymes. Thus, lysosomal cathepsins, acid phosphatase and various glycosidases have been used as diagnostic markers and to define metastatic potential in a range of cancers (Tappel, 2005). The underlying reason for this increase in lysosomal enzyme secretion may be linked to the increase in endosome-lysosome membrane recycling that is required to maintain plasma membrane area during rapid cell division (Boucrot & Kirchhausen, 2007). Increased lysosomal enzyme secretion has also been associated with extracellular matrix degradation and this can facilitate metastasis (Tayeb *et al*., 2005). The migration of metastatic cancer cells also involves upregulated exocytosis, as a means of membrane delivery to the leading edge of the migrating cell. This allows the formation of lamellipodia and filopodia, and thereby cellular movement. Exocytosis and cell division are both high energy demand cellular processes, and so it is not too surprising that autophagy has also been implicated in the

There is, however, controversy in the literature regarding the pro-survival and pro-death functions of autophagy (Hippert *et al*., 2006; Kundu & Thompson, 2008; Levine, 2007; Levine & Kroemer, 2008). The cyto-protective role that autophagy has under conditions of starvation or low energy supply, prevents apoptosis (Boya *et al*., 2005), and is therefore thought to promote cancer cell growth and survival within solid tumors prior to vascularization. In stark contrast, the suppression of autophagy via a number of regulatory pathways can lead to tumorigenesis (Gozuacik & Kimchi, 2006; Levine, 2007; Miracco *et al*., 2007; Pattingre *et al*., 2005). The increased tumorigenesis observed in *beclin1*/*Atg6* and *Atg5* murine mutants, and the high number of mono-allelic deletion mutations in these genes observed in different types of human cancer, indicate a direct tumor suppressor role for autophagy (Aita *et al*., 1999; Hippert *et al*., 2006; Kundu & Thompson, 2008; Levine, 2007). In addition, p53 and PTEN, which are frequently mutated in cancer patients, can stimulate autophagy (Bae *et al*., 2007; Lindmo *et al*., 2006; Shin *et al*., 2011; Wang *et al*., 2011); while PI3K, p38 MAPK and Akt, which are often activated in cancer, can suppress autophagy (Webber & Tooze, 2010). The apparent disparate roles of autophagy in cancer make it difficult to ascertain its exact function, and it also remains unclear whether exocytosis and autophagy are acting independently or as inter-linked

Cadwell *et al*., 2010; Fujita *et al*., 2008; Homer *et al*., 2010; Rioux, 2007; Saitoh *et al*., 2008).

synthesis and the maintenance of other cellular functions (He & Klionsky, 2009).

**4. Diseases that show links between exocytosis and autophagy** 

carcinogenic process, as a means of energy supply.

One of the upstream regulators, targets of rapamycin (TOR) acts as an inhibitor of autophagy. Inactivation of TOR leads to the assembly of an active Atg1 complex; Atg1:Atg13:Atg17 in yeast (Kamada *et al*., 2000; Nakatogawa *et al*., 2009), and Ulk1:Atg13:FIP200 in higher eukaryotes (Chang & Neufeld, 2010; Mehrpour *et al*., 2010). In the latter case, activation is thought to occur via a change in Atg13-mediated phosphorylation of Ulk1 (Chang & Neufeld, 2010), although the exact site and the induction signal for this initiation step remains unclear (Chang & Neufeld, 2009, 2010; Mehrpour *et al*., 2010).

The solution to the mystery of the origin of autophagic compartment may lay in the biology of Atg9, the only trans-membrane autophagic protein that is present throughout autophagosome maturation. In yeast, Atg9 has been observed to form clusters near the mitochondria, suggesting the possibility of a membrane pool for autophagy (Mari & Reggiori, 2010). However, an equivalent structure has yet to be identified in other eukaryotes, and there may be multiple nucleation sites, including the endoplasmic reticulum, Golgi, mitochondria (Hailey *et al*., 2010; Hamasaki & Yoshimori, 2010; Tooze & Yoshimori, 2010) and plasma membrane (Ravikumar *et al*., 2010). Recent studies in mammalian cells showed that Atg9 initially resides at the Golgi and is trafficked to recycling endosomes (Wang *et al*., 2011; Webber *et al*., 2007; Webber & Tooze, 2010). This suggests the involvement of the Golgi complex in the autophagic pathway. Starvation dependent trafficking of mammalian Atg9 to the pre-autophagosomal structure requires the Atg1/Ulk1 kinase, Atg13, as well as p38 MAPK interaction protein, p38IP (Webber & Tooze, 2010). Following Atg9 recruitment, nucleation of the pre-autophagosomal structure limiting membrane is controlled by a protein complex containing a member of the vacuolar protein sorting family, Vps34, and Atg6/Beclin1. Atg6 is crucial for the recruitment of other autophagic proteins to the pre-autophagosomal structure, while Vps34 kinase phosphorylates phosphatidylinositol (PI3P) in order to recruit Atg8 and Atg18 (Kundu & Thompson, 2008; Polson *et al*., 2010).

Two ubiquitin-like conjugation systems are required for the expansion and closure of the autophagosome, Atg5-Atg12 and the Atg8-phosphotidylethanolamine complex (Ichimura *et al*., 2000; Mizushima *et al*., 1998). In the first of these systems the conjugated form of Atg5- Atg12 associates with Atg16 dimers to become a multimeric Atg5-Atg12-Atg16 complex. It is believed that this Atg5-Atg12-Atg16 complex is required for the formation of preautophagosomal structures, and allows association with the second Atg8 related conjugation system. In the second ubiquitin-like conjugation system, cytosolic Atg8, or LC3 (microtubule-associated protein 1 light chain 3) in mammals, is modified by the attachment of the phospholipid anchor phosphatidylethanolamine, or PE. This step results in the localisation of Atg8/LC3-PE to the isolation membrane of the phagophore and may contribute to the expansion of autophagic membranes (McPhee & Baehrecke, 2009; Nakatogawa *et al*., 2007).

Once the autophagosome is closed by fusion of the expanding edges of the phagophore, its maturation proceeds through fusion with multi-vesicular bodies, late endosomes and lysosomes (Razi *et al*., 2009). It has been suggested that fusion of the autophagosome with endocytic compartments is facilitated by endosome membrane fusion machinery (Eskelinen, 2005) including the membrane targeting proteins Rab11 and Rab7 GTPases and membrane fusion protein complexes, such as SNAREs, ESCRT proteins, Vps28, Vps25, Vps32, Deep

One of the upstream regulators, targets of rapamycin (TOR) acts as an inhibitor of autophagy. Inactivation of TOR leads to the assembly of an active Atg1 complex; Atg1:Atg13:Atg17 in yeast (Kamada *et al*., 2000; Nakatogawa *et al*., 2009), and Ulk1:Atg13:FIP200 in higher eukaryotes (Chang & Neufeld, 2010; Mehrpour *et al*., 2010). In the latter case, activation is thought to occur via a change in Atg13-mediated phosphorylation of Ulk1 (Chang & Neufeld, 2010), although the exact site and the induction signal for this initiation step remains unclear (Chang & Neufeld, 2009, 2010; Mehrpour *et al*.,

The solution to the mystery of the origin of autophagic compartment may lay in the biology of Atg9, the only trans-membrane autophagic protein that is present throughout autophagosome maturation. In yeast, Atg9 has been observed to form clusters near the mitochondria, suggesting the possibility of a membrane pool for autophagy (Mari & Reggiori, 2010). However, an equivalent structure has yet to be identified in other eukaryotes, and there may be multiple nucleation sites, including the endoplasmic reticulum, Golgi, mitochondria (Hailey *et al*., 2010; Hamasaki & Yoshimori, 2010; Tooze & Yoshimori, 2010) and plasma membrane (Ravikumar *et al*., 2010). Recent studies in mammalian cells showed that Atg9 initially resides at the Golgi and is trafficked to recycling endosomes (Wang *et al*., 2011; Webber *et al*., 2007; Webber & Tooze, 2010). This suggests the involvement of the Golgi complex in the autophagic pathway. Starvation dependent trafficking of mammalian Atg9 to the pre-autophagosomal structure requires the Atg1/Ulk1 kinase, Atg13, as well as p38 MAPK interaction protein, p38IP (Webber & Tooze, 2010). Following Atg9 recruitment, nucleation of the pre-autophagosomal structure limiting membrane is controlled by a protein complex containing a member of the vacuolar protein sorting family, Vps34, and Atg6/Beclin1. Atg6 is crucial for the recruitment of other autophagic proteins to the pre-autophagosomal structure, while Vps34 kinase phosphorylates phosphatidylinositol (PI3P) in order to recruit Atg8 and Atg18 (Kundu &

Two ubiquitin-like conjugation systems are required for the expansion and closure of the autophagosome, Atg5-Atg12 and the Atg8-phosphotidylethanolamine complex (Ichimura *et al*., 2000; Mizushima *et al*., 1998). In the first of these systems the conjugated form of Atg5- Atg12 associates with Atg16 dimers to become a multimeric Atg5-Atg12-Atg16 complex. It is believed that this Atg5-Atg12-Atg16 complex is required for the formation of preautophagosomal structures, and allows association with the second Atg8 related conjugation system. In the second ubiquitin-like conjugation system, cytosolic Atg8, or LC3 (microtubule-associated protein 1 light chain 3) in mammals, is modified by the attachment of the phospholipid anchor phosphatidylethanolamine, or PE. This step results in the localisation of Atg8/LC3-PE to the isolation membrane of the phagophore and may contribute to the expansion of autophagic membranes (McPhee & Baehrecke, 2009;

Once the autophagosome is closed by fusion of the expanding edges of the phagophore, its maturation proceeds through fusion with multi-vesicular bodies, late endosomes and lysosomes (Razi *et al*., 2009). It has been suggested that fusion of the autophagosome with endocytic compartments is facilitated by endosome membrane fusion machinery (Eskelinen, 2005) including the membrane targeting proteins Rab11 and Rab7 GTPases and membrane fusion protein complexes, such as SNAREs, ESCRT proteins, Vps28, Vps25, Vps32, Deep

2010).

Thompson, 2008; Polson *et al*., 2010).

Nakatogawa *et al*., 2007).

Orange (Dor)/Vps18 and Carnation (Car)/Vps33a (Fader *et al*., 2008; Gutierrez *et al*., 2004a; Gutierrez *et al*., 2004b; Simonsen & Tooze, 2009), as well as the lysosomal membrane proteins, Lamp-1 and Lamp-2 (Tanaka *et al*., 2000). After fusion with the lysosome, degradation of protein, lipid, glycogen, RNA, DNA and other contents is dependent upon the action of lysosomal acid hydrolases (Koike *et al*., 2005; Tanaka *et al*., 2000; Tanida *et al*., 2005). The small molecules (e.g. amino acids and sugars) are then transported back to the cytosol for protein synthesis and the maintenance of other cellular functions (He & Klionsky, 2009).
