**Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay**

Chuan Hu, Nazarul Hasan and Krista Riggs *Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY* 

*USA* 

#### **1. Introduction**

228 Crosstalk and Integration of Membrane Trafficking Pathways

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Exocytosis is the fusion of transport vesicles with the plasma membrane. By exocytosis, eukaryotic cells secrete soluble proteins and endogenous chemicals to the extracellular space, and deliver new membrane proteins and lipids to the plasma membrane. A large body of work has demonstrated that the interactions of SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion events, including exocytosis (Bonifacino and Glick, 2004; Jahn et al., 2003; Rothman, 1994) (Fig. 1). The vesicle-associated membrane proteins (VAMPs), *i.e.*, VAMPs 1, 2, 3, 4, 5, 7 and 8, are v-SNAREs that reside in various post-Golgi vesicular compartments, and have been implicated in exocytosis. In this chapter, we review recent progress of using a novel cell fusion assay to analyze the specificity and membrane fusion activities of VAMPs (Hasan et al., 2010).

**Plasma membrane**

Fig. 1. Interactions of VAMPs and plasma membrane t-SNAREs drive exocytosis.

#### **2. SNAREs – Core machinery of vesicle fusion**

SNAREs are cytoplasmic oriented type I membrane proteins. SNAREs share one homologous domain, the 'SNARE motif,' which contains eight heptad repeats ready for coiled-coil formation. The SNARE proteins that mediate the fusion of synaptic vesicles with the presynaptic plasma membrane are well studied (Sollner et al., 1993b). In synapses, the v-

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay 231

membrane and intracellular vesicles (Zeng et al., 1998). In addition to vesicular transport from endosomes to lysosomes (Advani et al., 1999), the tetanus neurotoxin-insensitive VAMP (VAMP7) is involved in apical exocytosis in polarized epithelial cells (Galli et al., 1998; Pocard et al., 2007). Associated with early endosomes (Advani et al., 1998; Wong et al., 1998), VAMP8 (endobrevin) is required in regulated exocytosis in pancreatic acinar cells

VAMPs have high sequence homology in their SNARE motifs (Fig. 2). All VAMPs possess a conserved arginine residue at the center of SNARE motifs (Fig. 2), and have been classified as R-SNAREs based on crystal structures (Fasshauer et al., 1998). The N-terminal 51 residues of VAMP4 contain a dominant signal for targeting to the TGN, while the SNARE motif of VAMP5 is responsible for its targeting to the plasma membrane (Zeng et al., 2003). In VAMP7, the N-terminal 'longin domain' regulates subcellular targeting (Pryor et al., 2008). VAMPs 3, 4, 7 and 8 have broad tissue distribution (Advani et al., 1998; McMahon et al., 1993). Originally identified in nervous tissues, VAMPs 1 and 2 are also detected in skeletal muscle, fat and other tissues (Jagadish et al., 1996; Martin et al., 1998; Procino et al., 2008;

**VAMP1 ------------------------------------------------------------ VAMP2 ------------------------------------------------------------ VAMP3 ------------------------------------------------------------ VAMP4 ------------------------------------------------------------ VAMP5 ------------------------------------------------------------ VAMP7 MAILFAVVARGTTILAKHAWCGGNFLEVTEQILAKIPSENNKLTYSHGNYLFHYICQDRI 60 VAMP8 ------------------------------------------------------------** 

**VAMP1 ------------------------------MSAPAQPPAEGTEGTAPGGGPPGPPPNMTS 30 VAMP2 ------------------------------MSATAATAPP--AAPAGEGGPPAPPPNLTS 28 VAMP3 ------------------------------MST-------------------GPTAATGS 11 VAMP4 -------------MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFG 47 VAMP5 ---------------------------------------------------------MAG 3 VAMP7 VYLCITDDDFERSRAFNFLNEIKKRFQTTYGSRAQTALPYAMNSEFSSVLAAQLKHHSEN 120 VAMP8 -----------------------------------------------------MEEASEG 7 .** 

**VAMP1 --NRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKR 88 VAMP2 --NRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR 86 VAMP3 --NRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR 69 VAMP4 PRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRR 107 VAMP5 ---IELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQ 60 VAMP7 KGLDKVMETQAQVDELKGIMVRNIDLVAQRGERLELLIDKTENLVDSSVTFKTTSRNLAR 180 VAMP8 GGNDRVRNLQSEVEGVKNIMTQNVERILARGENLEHLRNKTEDLEATSEHFKTTSQKVAR 67 .: . \* :.: : :: \*. : \*. .\* \* :::: \* : \*.. : :: : VAMP1 KYWWKNCKMMIMLGAICAIIVVVIVRRD---------------------------- 116 VAMP2 KYWWKNLKMMIILGVICAIILIIIIVYFST-------------------------- 116 VAMP3 KYWWKNCKMWAIGITVLVIFIIIIIVWVVSS------------------------- 100 VAMP4 QMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT---------------------- 141 VAMP5 KKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN 116 VAMP7 AMCMKNLKLTIIIIIVSIVFIYIIVSPLCGGFTWPSCVKK---------------- 220 VAMP8 KFWWKNVKMIVLICVIVFIIILFIVLFATGAFS----------------------- 100** 

Fig. 2. Sequence alignment of human VAMP proteins. The conserved arginine residues in

**SNARE motifs**

 **.. : : .: .\*:** 

the center of SNARE motifs are labeled red.

(Wang et al., 2004).

SNARE VAMP2 resides in synaptic vesicles, whereas t-SNAREs syntaxin1 and synaptosomal-associated protein of 25 kD (SNAP-25) are located in the plasma membrane. Syntaxin1 and SNAP-25 constitute an acceptor complex for VAMP2 (Fasshauer and Margittai, 2004). The cytoplasmic domains of VAMP2, syntaxin1 and SNAP-25 form an extremely stable complex that is resistant to sodium dodecyl sulfate (SDS) (Hayashi et al., 1994) and heat stable up to ~90C (Yang et al., 1999), indicating that SNARE complex formation is thermodynamically favorable. One -helix from VAMP2, one -helix from syntaxin1 and two -helices from SNAP-25 intertwine to form a four-helix bundle (Sutton et al., 1998). Assembly of SNARE complexes is initiated at the N-termini and proceeds to the transmembrane domains at the C-termini in a zipper-like fashion (Stein et al., 2009). When v- and t-SNARE proteins are incorporated into liposomes, they spontaneously drive liposome fusion (McNew et al., 2000b; Weber et al., 1998), demonstrating that SNAREs form the minimal machinery for membrane fusion. Using a cell fusion assay, we showed that vand t-SNARE proteins ectopically expressed on the cell surface spontaneously drive cell-cell fusion (Hu et al., 2003; Hu et al., 2007), providing further proof that SNAREs form the core machinery for intracellular membrane fusion. After membrane fusion, the adapter protein SNAP (soluble NSF attachment protein) and the ATPase NSF (N-ethylmaleimide-sensitive factor) dissociate v-/t-SNARE complexes at the expense of ATP (Mayer et al., 1996; Sollner et al., 1993a) to free SNAREs for the next round of fusion.

Genomic analysis indicates that there are 36 SNAREs in humans (Bock et al., 2001). Individual members of the SNARE family localize to distinct subcellular organelles (Chen and Scheller, 2001), suggesting that each SNARE has a selective role in vesicle trafficking. Using yeast SNARE proteins as models, a series of experiments showed that to a remarkable degree the specificity of intracellular membrane fusion can be predicted from the pattern of liposome fusion mediated by isolated v- and t-SNARE proteins (McNew et al., 2000a; Parlati et al., 2002). However, membrane fusion by SNAREs in mammalian cells is more promiscuous (Brandhorst et al., 2006; Shen et al., 2007). Here we show that with the exception of VAMP5, VAMPs are essentially redundant in mediating membrane fusion with plasma membrane t-SNAREs (Hasan et al., 2010).

### **3. Roles of VAMPs in exocytosis**

VAMPs have been implicated in vesicle fusion with the plasma membrane, the *trans*-Golgi network (TGN) and endosomes. VAMP1 (synaptobrevin 1) and VAMP2 (synaptobrevin 2) mediate regulated exocytosis in neurons and endocrine cells (Hanson et al., 1997; Kesavan et al., 2007; Morgenthaler et al., 2003). In addition, VAMP2 is involved in the exocytosis of the water channel aquaporin 2 (Procino et al., 2008) and 51 integrin (Hasan and Hu, 2010), as well as insulin-stimulated translocation of the glucose transporter GLUT4 (Randhawa et al., 2000). Enriched in recycling endosomes and endosome-derived vesicles (Galli et al., 1994; McMahon et al., 1993), VAMP3 (cellubrevin) mediates the recycling of transferrin receptors to the cell surface (Galli et al., 1994), integrin trafficking (Luftman et al., 2009; Proux-Gillardeaux et al., 2005; Skalski and Coppolino, 2005), and the secretion of -granules in platelets (Feng et al., 2002; Polgar et al., 2002). Present primarily in the TGN, VAMP4 participates in the transport between the TGN and endosomes (Mallard et al., 2002; Steegmaier et al., 1999), as well as in homotypic fusion of early endosomes (Brandhorst et al., 2006). Expressed in muscle cells, VAMP5 (myobrevin) is associated with the plasma

SNARE VAMP2 resides in synaptic vesicles, whereas t-SNAREs syntaxin1 and synaptosomal-associated protein of 25 kD (SNAP-25) are located in the plasma membrane. Syntaxin1 and SNAP-25 constitute an acceptor complex for VAMP2 (Fasshauer and Margittai, 2004). The cytoplasmic domains of VAMP2, syntaxin1 and SNAP-25 form an extremely stable complex that is resistant to sodium dodecyl sulfate (SDS) (Hayashi et al., 1994) and heat stable up to ~90C (Yang et al., 1999), indicating that SNARE complex formation is thermodynamically favorable. One -helix from VAMP2, one -helix from syntaxin1 and two -helices from SNAP-25 intertwine to form a four-helix bundle (Sutton et al., 1998). Assembly of SNARE complexes is initiated at the N-termini and proceeds to the transmembrane domains at the C-termini in a zipper-like fashion (Stein et al., 2009). When v- and t-SNARE proteins are incorporated into liposomes, they spontaneously drive liposome fusion (McNew et al., 2000b; Weber et al., 1998), demonstrating that SNAREs form the minimal machinery for membrane fusion. Using a cell fusion assay, we showed that vand t-SNARE proteins ectopically expressed on the cell surface spontaneously drive cell-cell fusion (Hu et al., 2003; Hu et al., 2007), providing further proof that SNAREs form the core machinery for intracellular membrane fusion. After membrane fusion, the adapter protein SNAP (soluble NSF attachment protein) and the ATPase NSF (N-ethylmaleimide-sensitive factor) dissociate v-/t-SNARE complexes at the expense of ATP (Mayer et al., 1996; Sollner

Genomic analysis indicates that there are 36 SNAREs in humans (Bock et al., 2001). Individual members of the SNARE family localize to distinct subcellular organelles (Chen and Scheller, 2001), suggesting that each SNARE has a selective role in vesicle trafficking. Using yeast SNARE proteins as models, a series of experiments showed that to a remarkable degree the specificity of intracellular membrane fusion can be predicted from the pattern of liposome fusion mediated by isolated v- and t-SNARE proteins (McNew et al., 2000a; Parlati et al., 2002). However, membrane fusion by SNAREs in mammalian cells is more promiscuous (Brandhorst et al., 2006; Shen et al., 2007). Here we show that with the exception of VAMP5, VAMPs are essentially redundant in mediating membrane fusion with

VAMPs have been implicated in vesicle fusion with the plasma membrane, the *trans*-Golgi network (TGN) and endosomes. VAMP1 (synaptobrevin 1) and VAMP2 (synaptobrevin 2) mediate regulated exocytosis in neurons and endocrine cells (Hanson et al., 1997; Kesavan et al., 2007; Morgenthaler et al., 2003). In addition, VAMP2 is involved in the exocytosis of the water channel aquaporin 2 (Procino et al., 2008) and 51 integrin (Hasan and Hu, 2010), as well as insulin-stimulated translocation of the glucose transporter GLUT4 (Randhawa et al., 2000). Enriched in recycling endosomes and endosome-derived vesicles (Galli et al., 1994; McMahon et al., 1993), VAMP3 (cellubrevin) mediates the recycling of transferrin receptors to the cell surface (Galli et al., 1994), integrin trafficking (Luftman et al., 2009; Proux-Gillardeaux et al., 2005; Skalski and Coppolino, 2005), and the secretion of -granules in platelets (Feng et al., 2002; Polgar et al., 2002). Present primarily in the TGN, VAMP4 participates in the transport between the TGN and endosomes (Mallard et al., 2002; Steegmaier et al., 1999), as well as in homotypic fusion of early endosomes (Brandhorst et al., 2006). Expressed in muscle cells, VAMP5 (myobrevin) is associated with the plasma

et al., 1993a) to free SNAREs for the next round of fusion.

plasma membrane t-SNAREs (Hasan et al., 2010).

**3. Roles of VAMPs in exocytosis** 

membrane and intracellular vesicles (Zeng et al., 1998). In addition to vesicular transport from endosomes to lysosomes (Advani et al., 1999), the tetanus neurotoxin-insensitive VAMP (VAMP7) is involved in apical exocytosis in polarized epithelial cells (Galli et al., 1998; Pocard et al., 2007). Associated with early endosomes (Advani et al., 1998; Wong et al., 1998), VAMP8 (endobrevin) is required in regulated exocytosis in pancreatic acinar cells (Wang et al., 2004).

VAMPs have high sequence homology in their SNARE motifs (Fig. 2). All VAMPs possess a conserved arginine residue at the center of SNARE motifs (Fig. 2), and have been classified as R-SNAREs based on crystal structures (Fasshauer et al., 1998). The N-terminal 51 residues of VAMP4 contain a dominant signal for targeting to the TGN, while the SNARE motif of VAMP5 is responsible for its targeting to the plasma membrane (Zeng et al., 2003). In VAMP7, the N-terminal 'longin domain' regulates subcellular targeting (Pryor et al., 2008).

VAMPs 3, 4, 7 and 8 have broad tissue distribution (Advani et al., 1998; McMahon et al., 1993). Originally identified in nervous tissues, VAMPs 1 and 2 are also detected in skeletal muscle, fat and other tissues (Jagadish et al., 1996; Martin et al., 1998; Procino et al., 2008;


Fig. 2. Sequence alignment of human VAMP proteins. The conserved arginine residues in the center of SNARE motifs are labeled red.

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay 233

**FlippedSNAREs**

**Empty vector VAMP1**

**SS coiled-coil TMD**

**Fluorescence intensity**

**Empty vector VAMP5 <sup>B</sup>**

**Myc**

**A**

**Vector**

**VAMP1**

intensity of staining is obtained using CellQuest Pro software.

**VAMP3**

four hours after transfection with empty vector or flipped SNARE plasmids, unpermeabilized cells are stained with the anti-Myc antibody and analyzed by flow cytometry. (C) Representative FACS profiles of the cells transfected with empty vector or flipped VAMP1. (D) To express VAMPs and syntaxins at the same level at the cell surface, flipped SNARE plasmids are transfected at titrated concentrations. The mean fluorescence

**VAMP4**

Fig. 3. Expression of flipped SNAREs at the cell surface. (A) Domain structure of flipped SNAREs. (B) Twenty-four hours after transfection with empty vector pcDNA3.1(+) or flipped VAMP5 plasmid, unpermeabilized COS-7 cells are stained with an anti-Myc antibody. Representative confocal images are shown. Scale bar, 50 m. (C and D) Twenty-

**VAMP5**

**VAMP7**

**VAMP8**

**Syntaxin1**

**Syntaxin4**

**Counts**

**C**

**D**

**Mean fluorescence intensity**

Randhawa et al., 2000; Veale et al., 2010). Therefore, multiple VAMPs are co-expressed in mammalian cells. However, it is not clear if the seven VAMPs have differential membrane fusion activities. Using a cell fusion assay, we compare the membrane fusion activities of VAMPs.
