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

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; Cadwell *et al*., 2010; Fujita *et al*., 2008; Homer *et al*., 2010; Rioux, 2007; Saitoh *et al*., 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 carcinogenic process, as a means of energy supply.

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

At the Intersection of the Pathways for Exocytosis and Autophagy 117

Autophagy and exocytosis can have opposing or synergistic roles in cell function. For example, during times of reduced nutrient availability autophagy is stimulated, allowing cells to recycle cytoplasmic components, and exocytosis is reduced to conserve cellular constituents and energy (Shorer *et al*., 2005). In response to stimulation, specialised secretory cells (e.g. chromaffin neuroendicrine cells) divert energy utilization towards the exocytic pathway (Malacombe *et al*., 2006). Conversely, when a cell is presented with an immune challenge, both exocytosis and autophagy can be upregulated; exocytosis for the release of immune response factors and autophagy to clear invading pathogens from cells (Stow *et al*., 2009). Given these findings, it would appear to be of advantage to cells to have a mechanism

The dynamic flow of membrane and membrane proteins within a cell is mediated through the endosomal network (Figure 1). For example, lipids and proteins from the plasma membrane are recovered by the cell for cytosolic recycling via compensatory endocytosis, which also allows for the maintenance of membrane homeostasis at the sites of active exocytosis; directing endocytosed membrane back into the endosomal network or Golgi for degradation or recycling (Sramkova *et al*., 2009). This type of endocytosis is of particular importance in specialized secretory cells, such as, bladder umbrella cells (Khandelwal *et al*., 2008; Khandelwal *et al*., 2010), neurons (Kim & von Gersdorff, 2009; Llobet *et al*., 2011; Logiudice *et al*., 2009) and neuroendocrine cells (Engisch & Nowycky, 1998; Barg & Machado, 2008). This allows for the rapid recycling of secretory vesicles back into the reserve pool. Endosomes are at the nexus of the exocytic and autophagic pathways allowing for the sorting and directing of membrane. Thus, in yeast, Atg9 clusters are connected with both the endocytic and exocytic systems, and delivered to the phagophore assembly site via recycling endosomes (Geng *et al*., 2010; Mari *et al*., 2010). The recycling endosome's exocytic function is involved in the maintenance of cell polarity through the sorting of membrane proteins such as clathrin and cadherin (Farr *et al*., 2009). The recycling endosome machinery also plays a role in the fusion of multivesicular bodies with autophagosomes, which is an essential step in phagosome maturation (Fader & Colombo, 2009; Razi *et al*., 2009; Tooze & Razi, 2009). Recent studies have suggested a significant overlap of the molecular machinery used in these two biological processes (Bodemann *et al*., 2011; Geng *et al*., 2010). This involves the exocyst complex and its regulators (e.g. small GTPases), as well as membrane

**5.1 The endosomal network is involved in both exocytosis and autophagy** 

coordinating the activity of these two processes.

fusion machinery (e.g. SNAREs; Table 1).

(Fukai *et al*., 2003; Moskalenko *et al*., 2002).

**5.2.1 Ral small GTPase** 

**5.2 Small GTPases at the cross road of exocytosis and autophagy** 

Ras-like proteins (Ral) are small GTPases that function as an essential component of the cellular machinery regulating the post-Golgi targeting of exocytic vesicles to the plasma membrane (Balasubramanian *et al*., 2010; Chen *et al*., 2007; Kawato *et al*., 2008; Kim *et al*., 2010; Ljubicic *et al*., 2009; Lopez *et al*., 2008; Rondaij *et al*., 2008; Rosse *et al*., 2006; Shipitsin & Feig, 2004; Spiczka & Yeaman, 2008). Ral function is directly mediated by its interaction with the exocyst complex (Feig, 2003; Kawato *et al*., 2008; Mark *et al*., 1996; Mott *et al*., 2003), in particular Sec5 which has been shown to be essential for Ral-exocyst dependent exocytosis

processes in this disease. However, in one study, the trafficking of lysosomes in cancer cells was found to be linked to autophagosome formation through the common molecular machinery of the microtubule –dependent motor protein KIF5B (kinesin heavy chain protein 5B; Cardoso *et al*., 2009) a protein previously demonstrated to be involved in exocytosis (Varadi *et al*., 2002).

Neurodegenerative disorders including Parkinson's, Huntington's and Alzheimer's disease are progressive disorders, which have in common the loss of function of neurons in discrete areas of the central nervous system. This loss of function is thought to be a result of aggregation of misfolded proteins. Autophagy has a role in the degradation of misfolded protein (Yu *et al*., 2005), and the functional loss of *Atg5* or *Atg7* results in the accumulation of ubiquitinated protein aggregates, and a neurodegenerative phenotype (Hara, 2006; Komatsu *et al*., 2006). Furthermore, altered autophagy has been shown to be linked with altered exocytosis in a number of neurodegenerative disorders, leading to impaired release of neurotransmitters and increased inflammation (Gao & Hong, 2008; Keating, 2008). This highlights a direct link between autophagy and the recycling of the specialist secretory vesicles that control neurotransmission at the synaptic terminals of neurons.

Some inflammatory diseases, such as Crohn's disease, are thought to be caused by a breakdown in the regulation of exocytosis leading to increased secretion of proinflammatory factors (Barbier, 2003; Cadwell *et al*., 2010). In addition, a number of genetic screens of patients suffering from Crohn's disease have identified mutations in autophagy related genes (Barrett, 2008; Rioux, 2007). The autophagy related protein Atg16L is thought to function as a scaffold for LC3 lipidation, by dynamically localizing to the source of membrane involved in autophagosome formation (Fujita *et al*., 2008). A genetic defect in *Atg16L* may decrease the efficiency by which pathogens can be cleared from cells via autophagy, evoking an increased inflammatory response (Fujita *et al*., 2008; Homer *et al*., 2010; Saitoh *et al*., 2008). In addition, there is mounting evidence that defects in this autophagy gene can also lead to defects in exocytosis, causing a build-up of secretory granules in specific cell types (Cadwell *et al*., 2009). These concurrent defects in both exocytosis and autophagy may be one more piece of evidence for co-regulation and a shared molecular link between these two cellular processes, and raises the important question: is there common molecular machinery for exocytosis and autophagy?

#### **5. Exocytosis and autophagy: Common cellular functions and molecular machinery**

Exocytosis and autophagy are essential for a number of common biological processes, including; the immune response (Govind, 2008; Minty *et al*., 1983; Murray *et al*., 1998; Ostenson *et al*., 2006), cell growth (Brennwald & Rossi, 2007; Orlando & Guo, 2009; Wei & Zheng, 2011; Zhang *et al*., 2005), cell proliferation and apoptosis (Kundu, 2011; Shin *et al*., 2011; Zeng *et al*., 2012), and multicellular organism development (Gutnick *et al*., 2011; Hu *et al*., 2011; Sato & Sato, 2011; Tra *et al*., 2011). Autophagy and exocytosis both involve membrane trafficking and fusion events and so similar groups of molecular machinery may be required for both processes: such as GTPase proteins, that facilitate membrane tethering and SNARE proteins which are involved in membrane fusion. There is increasing evidence indicating shared molecular machinery between these processes, which provokes questions concerning possible dual regulation as a means of balance for these pathways.

processes in this disease. However, in one study, the trafficking of lysosomes in cancer cells was found to be linked to autophagosome formation through the common molecular machinery of the microtubule –dependent motor protein KIF5B (kinesin heavy chain protein 5B; Cardoso *et al*., 2009) a protein previously demonstrated to be involved in

Neurodegenerative disorders including Parkinson's, Huntington's and Alzheimer's disease are progressive disorders, which have in common the loss of function of neurons in discrete areas of the central nervous system. This loss of function is thought to be a result of aggregation of misfolded proteins. Autophagy has a role in the degradation of misfolded protein (Yu *et al*., 2005), and the functional loss of *Atg5* or *Atg7* results in the accumulation of ubiquitinated protein aggregates, and a neurodegenerative phenotype (Hara, 2006; Komatsu *et al*., 2006). Furthermore, altered autophagy has been shown to be linked with altered exocytosis in a number of neurodegenerative disorders, leading to impaired release of neurotransmitters and increased inflammation (Gao & Hong, 2008; Keating, 2008). This highlights a direct link between autophagy and the recycling of the specialist secretory

Some inflammatory diseases, such as Crohn's disease, are thought to be caused by a breakdown in the regulation of exocytosis leading to increased secretion of proinflammatory factors (Barbier, 2003; Cadwell *et al*., 2010). In addition, a number of genetic screens of patients suffering from Crohn's disease have identified mutations in autophagy related genes (Barrett, 2008; Rioux, 2007). The autophagy related protein Atg16L is thought to function as a scaffold for LC3 lipidation, by dynamically localizing to the source of membrane involved in autophagosome formation (Fujita *et al*., 2008). A genetic defect in *Atg16L* may decrease the efficiency by which pathogens can be cleared from cells via autophagy, evoking an increased inflammatory response (Fujita *et al*., 2008; Homer *et al*., 2010; Saitoh *et al*., 2008). In addition, there is mounting evidence that defects in this autophagy gene can also lead to defects in exocytosis, causing a build-up of secretory granules in specific cell types (Cadwell *et al*., 2009). These concurrent defects in both exocytosis and autophagy may be one more piece of evidence for co-regulation and a shared molecular link between these two cellular processes, and raises the important question: is

vesicles that control neurotransmission at the synaptic terminals of neurons.

there common molecular machinery for exocytosis and autophagy?

**5. Exocytosis and autophagy: Common cellular functions and molecular** 

concerning possible dual regulation as a means of balance for these pathways.

Exocytosis and autophagy are essential for a number of common biological processes, including; the immune response (Govind, 2008; Minty *et al*., 1983; Murray *et al*., 1998; Ostenson *et al*., 2006), cell growth (Brennwald & Rossi, 2007; Orlando & Guo, 2009; Wei & Zheng, 2011; Zhang *et al*., 2005), cell proliferation and apoptosis (Kundu, 2011; Shin *et al*., 2011; Zeng *et al*., 2012), and multicellular organism development (Gutnick *et al*., 2011; Hu *et al*., 2011; Sato & Sato, 2011; Tra *et al*., 2011). Autophagy and exocytosis both involve membrane trafficking and fusion events and so similar groups of molecular machinery may be required for both processes: such as GTPase proteins, that facilitate membrane tethering and SNARE proteins which are involved in membrane fusion. There is increasing evidence indicating shared molecular machinery between these processes, which provokes questions

exocytosis (Varadi *et al*., 2002).

**machinery** 

Autophagy and exocytosis can have opposing or synergistic roles in cell function. For example, during times of reduced nutrient availability autophagy is stimulated, allowing cells to recycle cytoplasmic components, and exocytosis is reduced to conserve cellular constituents and energy (Shorer *et al*., 2005). In response to stimulation, specialised secretory cells (e.g. chromaffin neuroendicrine cells) divert energy utilization towards the exocytic pathway (Malacombe *et al*., 2006). Conversely, when a cell is presented with an immune challenge, both exocytosis and autophagy can be upregulated; exocytosis for the release of immune response factors and autophagy to clear invading pathogens from cells (Stow *et al*., 2009). Given these findings, it would appear to be of advantage to cells to have a mechanism coordinating the activity of these two processes.

#### **5.1 The endosomal network is involved in both exocytosis and autophagy**

The dynamic flow of membrane and membrane proteins within a cell is mediated through the endosomal network (Figure 1). For example, lipids and proteins from the plasma membrane are recovered by the cell for cytosolic recycling via compensatory endocytosis, which also allows for the maintenance of membrane homeostasis at the sites of active exocytosis; directing endocytosed membrane back into the endosomal network or Golgi for degradation or recycling (Sramkova *et al*., 2009). This type of endocytosis is of particular importance in specialized secretory cells, such as, bladder umbrella cells (Khandelwal *et al*., 2008; Khandelwal *et al*., 2010), neurons (Kim & von Gersdorff, 2009; Llobet *et al*., 2011; Logiudice *et al*., 2009) and neuroendocrine cells (Engisch & Nowycky, 1998; Barg & Machado, 2008). This allows for the rapid recycling of secretory vesicles back into the reserve pool. Endosomes are at the nexus of the exocytic and autophagic pathways allowing for the sorting and directing of membrane. Thus, in yeast, Atg9 clusters are connected with both the endocytic and exocytic systems, and delivered to the phagophore assembly site via recycling endosomes (Geng *et al*., 2010; Mari *et al*., 2010). The recycling endosome's exocytic function is involved in the maintenance of cell polarity through the sorting of membrane proteins such as clathrin and cadherin (Farr *et al*., 2009). The recycling endosome machinery also plays a role in the fusion of multivesicular bodies with autophagosomes, which is an essential step in phagosome maturation (Fader & Colombo, 2009; Razi *et al*., 2009; Tooze & Razi, 2009). Recent studies have suggested a significant overlap of the molecular machinery used in these two biological processes (Bodemann *et al*., 2011; Geng *et al*., 2010). This involves the exocyst complex and its regulators (e.g. small GTPases), as well as membrane fusion machinery (e.g. SNAREs; Table 1).

#### **5.2 Small GTPases at the cross road of exocytosis and autophagy**

#### **5.2.1 Ral small GTPase**

Ras-like proteins (Ral) are small GTPases that function as an essential component of the cellular machinery regulating the post-Golgi targeting of exocytic vesicles to the plasma membrane (Balasubramanian *et al*., 2010; Chen *et al*., 2007; Kawato *et al*., 2008; Kim *et al*., 2010; Ljubicic *et al*., 2009; Lopez *et al*., 2008; Rondaij *et al*., 2008; Rosse *et al*., 2006; Shipitsin & Feig, 2004; Spiczka & Yeaman, 2008). Ral function is directly mediated by its interaction with the exocyst complex (Feig, 2003; Kawato *et al*., 2008; Mark *et al*., 1996; Mott *et al*., 2003), in particular Sec5 which has been shown to be essential for Ral-exocyst dependent exocytosis (Fukai *et al*., 2003; Moskalenko *et al*., 2002).

At the Intersection of the Pathways for Exocytosis and Autophagy 119

**Protein Role in Exocytosis References Role in Autophagy References** 

Involved in lysosome fusion

autophagosome maturation

Transmembrane protein required for the transport and assembly of membrane during autophagosome formation

Functions as a scaffold for LC3 lipidation, required

autophagosome formation

during

(Fader *et al*., 2009)

(He *et al*., 2009)

(Fujita *et al*., 2008)

during

(Galli *et al*., 1998; Oishi *et al*.,

(Bruns *et al*., 2011; Mari *et al*.,

(Cadwell *et al*., 2008; Cadwell *et* 

In addition to its well documented role in exocytosis, recent evidence from mammalian cell cultures indicates that RalB is involved in the formation of autophagosomes (Bodemann *et al*., 2011). The crucial role for RalB as an upstream activator of autophagy is illustrated by the fact that the over-expression of its active GTP-bound form was sufficient to induce autophagy, even in the absence of autophagy-specific stimuli (Bodemann *et al*., 2011). RalB is present on sites of nascent autophagosome formation, together with Beclin1 and Atg5, and its depletion, similar to the depletion of Atg5 and Beclin1, significantly impaired the formation of starvation-induced LC3/Atg8 punctae and the turnover of LC3/Agt8. Interestingly, depletion of RalB also impaired the digestion of autophagocytosed *Salmonella typhimurium*. The autophagy-related function of RalB appears to be mediated by its effector Exo84, a component of the exocyst complex (Bodemann *et al*., 2011). Activated by starvation, RalB triggers Exo84 interaction with the autophagy initiation component Beclin1. Intriguingly, the alternative RalB roles in exocytosis and autophagy appear to be driven by environmental signal/s, as nutrient availability determines the RalB coupling preferences to a down-stream effector; endogenous RalB preferentially associates with Exo84 in nutrient poor conditions and with Sec5 under nutrient rich conditions (Bodemann *et al*., 2011). This model has not been investigated in higher eukaryotes, but these findings in yeast suggest a role for the exocyst complex as a scaffold for the assembly of a number of important

The yeast Rab GTPase Sec4 and its activator Sec2 have well-established roles in the tethering of secretory vesicles to sites of active exocytosis, in a process mediated by interaction with the exocyst complex component Sec15 (Geng *et al*., 2010) . Recent studies indicate that Sec2 and Sec4 also have a role in anterograde trafficking of the autophagic membrane protein Atg9, as silencing of Sec4 blocked the delivery of Atg9 to the pre-autophagosomal structure

2006)

2010)

*al*., 2009)

Table 1. Proteins involved in both autophagy and exocytosis

**VAMP7** Involved in

**Atg16L** Involved in

autophagy initiators.

**5.2.2 Yeast Rab small GTPase Sec4** 

constitutive exocytosis in a number of cell

has been found on secretory vesicles. May have a role in unconventional secretion

secretion from secretory granules in intestinal Paneth cells

types

**Atg9** Unknown role but


RalB but not RalA involved in initation of autophagy in mammalian cell lines. Over expression of active RalB enhances autophagy while depletion decreases autophagy.

Facilitates fusion of the autophagosome with endocytic compartments.

recruitment of Atg9 to the PAS.

Proposed as a scaffold for the initiation of autophagy complexes.

Involved in the formation of Atg9 associated tubulevesicular clusters emanating from the

PAS

(Bodemann *et al*., 2011)

(Fader *et al*., 2008)

(Geng *et al*., 2010)

(Bodemann *et al*., 2011; Farré & Subramani, 2011)

(Nair *et al*., 2011)

**Protein Role in Exocytosis References Role in Autophagy References** 

(Balasubramanian *et al*., 2010; Brymora *et al*., 2001; Chen *et al*., 2011a; Chen *et al*., 2007; Fukai *et al*., 2003; Kawato *et al*., 2008; Li *et al*., 2007; Ljubicic *et al*., 2009; Lopez *et al*., 2008; Mark *et al*., 1996; Moskalenko *et al*., 2002; Mott *et al*., 2003; Shipitsin & Feig,

(Langevin *et al*., 2005; Oztan *et al*., 2007; Shandala *et al*., 2011; Ward *et al*., 2005; Wu *et al*., 2005;

(Guo *et al*., 1999) Involved in the

(He *et al*., 2007; He & Guo, 2009; Jin *et al*., 2011; Langevin *et al*., 2005; Morgera *et al*., 2012)

(Aalto *et al*., 1993; Brennwald *et* 

*al*., 1994)

Zhang *et al*., 2004)

2004)

**Ral** Interacts with

**Rab11** Bound to exocytic

exocyst

**Sec4** Allows the

**Exocyst Complex**  component Sec15 to assist tethering of vesicles to the plasma membrane.

interaction of the secretory vesicle with the exocyst complex via Sec15 to facilitate tethering to the plasma membrane.

Octomeric complex required for tethering of exocytic vesicles to the plasma membrane in a site specific manner

denotes the site of exocytosis on the plasma membrane, possibly through interactions with the exocyst complex and its effectors

**Sso1/2-Sec9** t-SNARE that

vesicles and is involved in the anterograde trafficking of vesicles from recycling endosomes to the plasma membrane. Interacts with the

exocyst via Sec5 to facilitate the tethering of vesicles to the plasma membrane.


Table 1. Proteins involved in both autophagy and exocytosis

In addition to its well documented role in exocytosis, recent evidence from mammalian cell cultures indicates that RalB is involved in the formation of autophagosomes (Bodemann *et al*., 2011). The crucial role for RalB as an upstream activator of autophagy is illustrated by the fact that the over-expression of its active GTP-bound form was sufficient to induce autophagy, even in the absence of autophagy-specific stimuli (Bodemann *et al*., 2011). RalB is present on sites of nascent autophagosome formation, together with Beclin1 and Atg5, and its depletion, similar to the depletion of Atg5 and Beclin1, significantly impaired the formation of starvation-induced LC3/Atg8 punctae and the turnover of LC3/Agt8. Interestingly, depletion of RalB also impaired the digestion of autophagocytosed *Salmonella typhimurium*. The autophagy-related function of RalB appears to be mediated by its effector Exo84, a component of the exocyst complex (Bodemann *et al*., 2011). Activated by starvation, RalB triggers Exo84 interaction with the autophagy initiation component Beclin1. Intriguingly, the alternative RalB roles in exocytosis and autophagy appear to be driven by environmental signal/s, as nutrient availability determines the RalB coupling preferences to a down-stream effector; endogenous RalB preferentially associates with Exo84 in nutrient poor conditions and with Sec5 under nutrient rich conditions (Bodemann *et al*., 2011). This model has not been investigated in higher eukaryotes, but these findings in yeast suggest a role for the exocyst complex as a scaffold for the assembly of a number of important autophagy initiators.

#### **5.2.2 Yeast Rab small GTPase Sec4**

The yeast Rab GTPase Sec4 and its activator Sec2 have well-established roles in the tethering of secretory vesicles to sites of active exocytosis, in a process mediated by interaction with the exocyst complex component Sec15 (Geng *et al*., 2010) . Recent studies indicate that Sec2 and Sec4 also have a role in anterograde trafficking of the autophagic membrane protein Atg9, as silencing of Sec4 blocked the delivery of Atg9 to the pre-autophagosomal structure

At the Intersection of the Pathways for Exocytosis and Autophagy 121

in cells depleted for core exocyst components. For example, the depletion of Sec8 rendered cells insensitive to starvation stimulation, and impaired autophagy to the same extent as seen for the depletion of Atg5 and Beclin1 (Bodemann *et al*., 2011). Further interrogation of this system showed that the localization of exocyst components, with the autophagy initiator Atg1/Ulk1 and other proteins involved in isolation membrane formation, was altered following the induction of autophagy (Bodemann *et al*., 2011). As these processes are under the control of Ral or Rab, the differential recruitment of the exocyst to the target

membrane might depend on the signals upstream of these small GTPases.

2010; Nair *et al*., 2011) .

membranes?

**5.5 The role of autophagy genes in secretion** 

**5.4 SNARE proteins and membrane fusion during exocytosis and autophagy** 

Both autophagosome maturation and anterograde vesicle trafficking via the exocytic route involve a series of membrane fusion steps, the execution of which is controlled by SNAREs. Recent studies in yeast have indicated that some exocytic t-SNAREs may also play a role in membrane dynamics during autophagy (Geng *et al*., 2010; Nair *et al*., 2011). The anterograde trafficking of the key autophagic membrane determinant, Atg9, depends on interaction with exocytic Sso1-Sec9, as well as on the endosomal t-SNARE Tlg2 and the v-SNAREs Sec22 and Ykt6. Sso1/2 and Sec9 SNAREs are also responsible for the formation of the Atg9 associated tubular-vesicular clusters emanating from the pre-autophagosomal structure, and their depletion results in Atg9 localization to small vesicular structures, possibly *trans*-Golgi network derived secretory vesicles, that fail to be delivered to the pre-autophagosomal structure (Nair *et al*., 2011). This failure of Atg9 delivery to pre-autophagosomal structure abolishes Atg8 recruitment, and thereby abrogates autophagosome biogenesis (Geng *et al*.,

Another group of SNAREs, the vesicle-associated membrane proteins (VAMPs), appear to be involved in membrane fusion events in both autophagy and exocytosis. One the one hand, during autophagosome maturation, it has been shown that VAMP3 and VAMP7 are required for sequential fusion with multivesicular bodies and lysosomes respectively (Fader *et al*., 2009). In HeLa cells, VAMP7 has been shown to be involved in homotypic fusion of Atg16L1 positive vesicles to allow autophagosome biogenesis (Moreau *et al*., 2011). On the other hand, it has been recently demonstrated that VAMP7 is involved in constitutive exocytosis in HSY cells (Oishi *et al*., 2006), and in apical trafficking of exocytic vesicles in polarized epithelial cells, such as MDCK cells and CaCo-2 cells (Galli *et al*., 1998). VAMP3 has been postulated to be a v-SNARE for early and recycling endosomes, with a role in constitutive exocytosis, but its role might be redundant as mice with a null mutation for this gene were normal in most endocytic and exocytic pathways, including constitutive exocytosis (Wang *et al*., 2004; Yang *et al*., 2001). The question remains whether these functions of SNAREs are restricted to specific tissues, or universal for all tissues, and if so, what are the upstream signals that direct these SNAREs to either the exocytic or autophagic

There is emerging evidence of involvement of autophagy proteins in polarized secretion involving lysosomes. In bone resorptive osteoclasts, Atg5, Atg7, Atg4B, and LC3/Atg8 participate in directing lysosomes to fuse with the plasma membrane and in the release of the lytic enzyme cathepsin K into the extracellular space (DeSelm *et al*., 2011). This type of

(Geng *et al*., 2010). Furthermore, when the domain of Sec4 that is known to interact with Sec15 was altered, the effect on autophagy was equivalent to the effect of Sec4 silencing. Taking into account that there is no apparent role for Sec15 in autophagy, this suggests that autophagy-specific proteins may compete for this Sec15-binding domain in order to switch the function of activated GTP-bound Sec4 between exocytosis and autophagy.

## **5.2.3 Metazoan Rab11 small GTPase**

Rab11 is a small GTPase, which is most often referred to as a recycling endosome marker. However, it has also been observed on vesicles bound for exocytosis (Shandala *et al*., 2011; Ward *et al*., 2005), and amphisomes; an intermediate compartment that is formed during autophagosome maturation, prior to lysosomal fusion (Fader & Colombo, 2009)

The exocytic role for Rab11 is mediated by its association with the Sec15 exocyst component. This has been shown in MSCK cells (Oztan *et al*., 2007; Zhang *et al*., 2004), and in *Drosophila* photoreceptor and sensory neuron cells (Wu *et al*., 2005). Rab11 is important for the anterograde trafficking of; numerous membrane receptors (Chernyshova *et al*., 2011), the epithelial sodium channel complex of the cortical collecting duct of the kidneys (Butterworth *et al*., 2012), and DE-Cadherin in polarised cells (Langevin *et al*., 2005; Wu *et al*., 2005; Zhang *et al*., 2004), as well as the calcium dependent exocytosis of growth hormones (Ren *et al*., 1998; Takaya *et al*., 2007). A number of intracellular pathogens, such as *Porphyromonas gingivalis*, influenza A and HIV, have been reported to hi-jack Rab11 dependent anterograde trafficking as a means of escape from host cells (Kadiu & Gendelman, 2011; Momose *et al*., 2011; Takeuchi *et al*., 2011).

An example of coordinated exocytosis and autophagy comes from the biology of multivesicular bodies (MVBs). MVBs are specialised late endosomes, a crucial intermediate in the internalization of nutrients, ligands and receptors into small intraluminal vesicles, also known as exosomes (Fader & Colombo, 2009). Rab11 decorates MVBs and is involved in both the biogenesis of MVBs and exosome release (Fader *et al*., 2008). During the maturation of hematopoietic progenitors into reticulocytes and erythrocytes, proteins that are not required at the mature stage are sequestered into exosomes of MVBs. In this scenario Rab11 is involved in the targeting of MVBs to the plasma membrane, where exosomes are released into the extracellular milieu (Fader & Colombo, 2006). Active Rab11 is also required for the interaction of MVBs with autophagosomes, where the resulting calcium-stimulated fusion of these organelles promotes efficient degradation of autophagic contents (Fader *et al*., 2008; Savina *et al*., 2005). Thus, Rab11 may represent a critical regulator of membrane flow between recycling endosomes (as a source of exocytic vesicles) and multivesicular bodies, where it can be engaged in both autophagic maturation and secretion.

#### **5.3 The exocyst and the initiation of autophagy**

Components of the exocyst complex involved in regulated and polarized exocytosis have also been shown to associate with a number of essential autophagy proteins (Bodemann *et al*., 2011). Exocyst components Sec3 and Sec8 interact *in vitro* with positive (FIP200, ATG14L) and negative (RUBICON) regulators of autophagy, as well as with the phagophore expansion complex Atg5-Atg12 (Bodemann *et al*., 2011). The functionality of these physical interactions is confirmed by the fact that LC3/Atg8 autophagosome formation was impaired

(Geng *et al*., 2010). Furthermore, when the domain of Sec4 that is known to interact with Sec15 was altered, the effect on autophagy was equivalent to the effect of Sec4 silencing. Taking into account that there is no apparent role for Sec15 in autophagy, this suggests that autophagy-specific proteins may compete for this Sec15-binding domain in order to switch

Rab11 is a small GTPase, which is most often referred to as a recycling endosome marker. However, it has also been observed on vesicles bound for exocytosis (Shandala *et al*., 2011; Ward *et al*., 2005), and amphisomes; an intermediate compartment that is formed during

The exocytic role for Rab11 is mediated by its association with the Sec15 exocyst component. This has been shown in MSCK cells (Oztan *et al*., 2007; Zhang *et al*., 2004), and in *Drosophila* photoreceptor and sensory neuron cells (Wu *et al*., 2005). Rab11 is important for the anterograde trafficking of; numerous membrane receptors (Chernyshova *et al*., 2011), the epithelial sodium channel complex of the cortical collecting duct of the kidneys (Butterworth *et al*., 2012), and DE-Cadherin in polarised cells (Langevin *et al*., 2005; Wu *et al*., 2005; Zhang *et al*., 2004), as well as the calcium dependent exocytosis of growth hormones (Ren *et al*., 1998; Takaya *et al*., 2007). A number of intracellular pathogens, such as *Porphyromonas gingivalis*, influenza A and HIV, have been reported to hi-jack Rab11 dependent anterograde trafficking as a means of escape from host cells (Kadiu & Gendelman, 2011; Momose *et al*., 2011; Takeuchi

An example of coordinated exocytosis and autophagy comes from the biology of multivesicular bodies (MVBs). MVBs are specialised late endosomes, a crucial intermediate in the internalization of nutrients, ligands and receptors into small intraluminal vesicles, also known as exosomes (Fader & Colombo, 2009). Rab11 decorates MVBs and is involved in both the biogenesis of MVBs and exosome release (Fader *et al*., 2008). During the maturation of hematopoietic progenitors into reticulocytes and erythrocytes, proteins that are not required at the mature stage are sequestered into exosomes of MVBs. In this scenario Rab11 is involved in the targeting of MVBs to the plasma membrane, where exosomes are released into the extracellular milieu (Fader & Colombo, 2006). Active Rab11 is also required for the interaction of MVBs with autophagosomes, where the resulting calcium-stimulated fusion of these organelles promotes efficient degradation of autophagic contents (Fader *et al*., 2008; Savina *et al*., 2005). Thus, Rab11 may represent a critical regulator of membrane flow between recycling endosomes (as a source of exocytic vesicles) and multivesicular

bodies, where it can be engaged in both autophagic maturation and secretion.

Components of the exocyst complex involved in regulated and polarized exocytosis have also been shown to associate with a number of essential autophagy proteins (Bodemann *et al*., 2011). Exocyst components Sec3 and Sec8 interact *in vitro* with positive (FIP200, ATG14L) and negative (RUBICON) regulators of autophagy, as well as with the phagophore expansion complex Atg5-Atg12 (Bodemann *et al*., 2011). The functionality of these physical interactions is confirmed by the fact that LC3/Atg8 autophagosome formation was impaired

**5.3 The exocyst and the initiation of autophagy** 

the function of activated GTP-bound Sec4 between exocytosis and autophagy.

autophagosome maturation, prior to lysosomal fusion (Fader & Colombo, 2009)

**5.2.3 Metazoan Rab11 small GTPase** 

*et al*., 2011).

in cells depleted for core exocyst components. For example, the depletion of Sec8 rendered cells insensitive to starvation stimulation, and impaired autophagy to the same extent as seen for the depletion of Atg5 and Beclin1 (Bodemann *et al*., 2011). Further interrogation of this system showed that the localization of exocyst components, with the autophagy initiator Atg1/Ulk1 and other proteins involved in isolation membrane formation, was altered following the induction of autophagy (Bodemann *et al*., 2011). As these processes are under the control of Ral or Rab, the differential recruitment of the exocyst to the target membrane might depend on the signals upstream of these small GTPases.

#### **5.4 SNARE proteins and membrane fusion during exocytosis and autophagy**

Both autophagosome maturation and anterograde vesicle trafficking via the exocytic route involve a series of membrane fusion steps, the execution of which is controlled by SNAREs. Recent studies in yeast have indicated that some exocytic t-SNAREs may also play a role in membrane dynamics during autophagy (Geng *et al*., 2010; Nair *et al*., 2011). The anterograde trafficking of the key autophagic membrane determinant, Atg9, depends on interaction with exocytic Sso1-Sec9, as well as on the endosomal t-SNARE Tlg2 and the v-SNAREs Sec22 and Ykt6. Sso1/2 and Sec9 SNAREs are also responsible for the formation of the Atg9 associated tubular-vesicular clusters emanating from the pre-autophagosomal structure, and their depletion results in Atg9 localization to small vesicular structures, possibly *trans*-Golgi network derived secretory vesicles, that fail to be delivered to the pre-autophagosomal structure (Nair *et al*., 2011). This failure of Atg9 delivery to pre-autophagosomal structure abolishes Atg8 recruitment, and thereby abrogates autophagosome biogenesis (Geng *et al*., 2010; Nair *et al*., 2011) .

Another group of SNAREs, the vesicle-associated membrane proteins (VAMPs), appear to be involved in membrane fusion events in both autophagy and exocytosis. One the one hand, during autophagosome maturation, it has been shown that VAMP3 and VAMP7 are required for sequential fusion with multivesicular bodies and lysosomes respectively (Fader *et al*., 2009). In HeLa cells, VAMP7 has been shown to be involved in homotypic fusion of Atg16L1 positive vesicles to allow autophagosome biogenesis (Moreau *et al*., 2011). On the other hand, it has been recently demonstrated that VAMP7 is involved in constitutive exocytosis in HSY cells (Oishi *et al*., 2006), and in apical trafficking of exocytic vesicles in polarized epithelial cells, such as MDCK cells and CaCo-2 cells (Galli *et al*., 1998). VAMP3 has been postulated to be a v-SNARE for early and recycling endosomes, with a role in constitutive exocytosis, but its role might be redundant as mice with a null mutation for this gene were normal in most endocytic and exocytic pathways, including constitutive exocytosis (Wang *et al*., 2004; Yang *et al*., 2001). The question remains whether these functions of SNAREs are restricted to specific tissues, or universal for all tissues, and if so, what are the upstream signals that direct these SNAREs to either the exocytic or autophagic membranes?

#### **5.5 The role of autophagy genes in secretion**

There is emerging evidence of involvement of autophagy proteins in polarized secretion involving lysosomes. In bone resorptive osteoclasts, Atg5, Atg7, Atg4B, and LC3/Atg8 participate in directing lysosomes to fuse with the plasma membrane and in the release of the lytic enzyme cathepsin K into the extracellular space (DeSelm *et al*., 2011). This type of

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