**4. Membrane fusion by VAMPs and plasma membrane t-SNAREs**

#### **4.1 Fusogenic interactions of VAMPs and plasma membrane t-SNAREs**

Using the enzymatic fusion assay (Fig. 4B), we examine the fusogenic pairings between the seven VAMPs and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 (Fasshauer and Margittai, 2004) and syntaxin4/SNAP-25 (Reed et al., 1999). Robust βgalactosidase expression is detected when VAMPs 1, 2, 3, 4, 7 or 8 are combined with syntaxin1/SNAP-25 (Fig. 5A) or syntaxin4/SNAP-25 (Fig. 5B), indicating that these VAMPs mediate membrane fusion with plasma membrane t-SNAREs. With syntaxin1/SNAP-25, the

6.7 g/ml tunicamycin and 0.67 mM DTT. v-Cells (1.2 × 105) are added to each coverslip already containing the t-cells. After 24 hours at 37C in 5% CO2, the cells are gently washed once with PBS++, then fixed with 4% paraformaldehyde. Confocal images are collected on an Olympus laser scanning confocal microscope. The 488 nm argon laser line is used to excite EGFP and the 543 nm HeNe laser line is used to excite DsRed2. To prevent crosscontamination between EGFP and DsRed2, each channel is imaged sequentially before

The day before transfection, 1.2 × 106 COS-7 cells are seeded in each 100-mm cell culture dish, and 2 × 105 COS-7 cells are seeded in each well of 6-well plates. For v-cells, 5 g each of flipped v-SNARE plasmid are cotransfected with 5 µg of pTet-Off into the cells in each 100 mm culture dish. Control cells are cotransfected with empty vector pcDNA3.1(+) and pTet-Off. For t-cells, 1 µg each of flipped syntaxins 1 or 4, SNAP-25 and pBI-G are cotransfected into the cells in each well of the 6-well plates. There are putative N-glycosylation motifs (Asn-X-Ser/Thr) in VAMPs. Our previous studies (Hu et al., 2003; Hu et al., 2007) showed that N-glycosylation disrupts the function of flipped SNAREs, and that treatment of tunicamycin, an antibiotic that inhibits N-glycosylation (Elbein, 1984), restores the fusion activities of flipped SNAREs. To prevent N-glycosylation of VAMPs 1, 4, 5, 7 and 8, v-cells expressing these VAMP proteins and control cells are incubated in cell culture medium containing 10 g/ml of tunicamycin during transfection. Since we have mutated the putative N-glycosylation sites in flipped VAMP2 (Hu et al., 2003) and VAMP3 proteins (Hu et al., 2007), v-cells that express VAMPs 2 or 3 are not treated with tunicamycin. Since flipped syntaxins 1 or 4, SNAP-25 proteins are not N-glycosylated (Hu et al., 2003; Hu et al.,

Twenty-four hours after transfection, the v-cells are detached from the culture dishes with Enzyme-free Cell Dissociation Buffer, and added (4.8 × 105 cells) to each well already containing the t-cells. After 6, 12 or 24 hours at 37C in 5% CO2, the expression of βgalactosidase is measured using the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer according to the manufacturer's instructions (Promega). The cells are washed twice with PBS, and then lysed in Reporter Lysis Buffer. Cell lysates are mixed with equal volume of Assay 2× Buffer. As a blank control, Reporter Lysis Buffer is mixed with Assay 2× Buffer. After 90 minutes, the colorimetric reaction is stopped by adding 1 M sodium carbonate, and absorbance at 420 nm is measured using a HITACHI 100-40

Using the enzymatic fusion assay (Fig. 4B), we examine the fusogenic pairings between the seven VAMPs and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 (Fasshauer and Margittai, 2004) and syntaxin4/SNAP-25 (Reed et al., 1999). Robust βgalactosidase expression is detected when VAMPs 1, 2, 3, 4, 7 or 8 are combined with syntaxin1/SNAP-25 (Fig. 5A) or syntaxin4/SNAP-25 (Fig. 5B), indicating that these VAMPs mediate membrane fusion with plasma membrane t-SNAREs. With syntaxin1/SNAP-25, the

**4. Membrane fusion by VAMPs and plasma membrane t-SNAREs 4.1 Fusogenic interactions of VAMPs and plasma membrane t-SNAREs** 

merging the images.

spectrophotometer.

**3.4.4 Enzymatic cell fusion assay** 

2007), the t-cells are not treated with tunicamycin.

six VAMPs drive fusion to a similar degree. With syntaxin4/SNAP-25, VAMP8 fuses less efficiently than VAMPs 1, 2, 3 and 4 (31% lower fusion activity and *P* = 0.046 vs. VAMP1, Fig. 5B). In contrast, when VAMP5 is combined with the t-SNAREs, only baseline βgalactosidase activity is detected (Figs. 5A and B), suggesting that VAMP5 does not drive membrane fusion with the t-SNAREs. The stronger fusion activities of syntaxin4/SNAP-25 than syntaxin1/SNAP-25 (compare Figs. 5A and B) are likely caused by higher cell surface expression of syntaxin4/SNAP-25 than syntaxin1/SNAP-25 and higher fusion activity of syntaxin4 than syntaxin1 (Hasan et al., 2010). Together, the data shown in Fig. 5 indicate that VAMPs 1, 2, 3, 4, 7 and 8, but not VAMP5, drive membrane fusion when partnering with plasma membrane t-SNAREs.

Fig. 5. Cell fusion by VAMPs and plasma membrane t-SNAREs. Twenty-four hours after combining v-cells that express VAMPs 1, 2, 3, 4, 5, 7 or 8 and t-cells that express (A) syntaxin1/SNAP-25 or (B) syntaxin4/SNAP-25, cell fusion is quantified using the enzymatic cell fusion assay. Control cells (-VAMP) are transfected with the empty vector. The flipped SNARE plasmids are transfected at the same concentration. Error bars represent standard deviation of three independent experiments. \* *P* < 0.05 vs. VAMP1.

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay 239

VAMPs 1 and 3 have comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 have 36%, 26% and 54% lower fusion activities, respectively (Fig. 6B). As expected, only baseline β-galactosidase activity is detected when VAMP5 is paired with the t-SNAREs. These data indicate that VAMPs have differential membrane fusion activities with plasma membrane t-SNAREs. However, when expressed at higher levels, VAMP4 drives membrane fusion as efficiently as VAMPs 1 and 3 (Fig. 5). Since the differences of fusion activities among the fusogenic VAMPs are within a factor of 2, these results imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating membrane fusion with

**VAMP 1 3 4 5 78-**

**t-SNARE Syntaxin4/SNAP-25**

**VAMP 1 3 4 5 78 -**

Fig. 6. Comparison of fusion activities of VAMPs. With optimized transfection conditions,

syntaxin1/SNAP-25 (A) and syntaxin4/SNAP-25 (B) are expressed at the same level. Cell fusion is quantified using the enzymatic fusion assay. The fusion activities (OD420) of control cells (-VAMP) and the v-cells expressing VAMPs 3, 4, 5, 7 or 8 are normalized to the fusion activity of the v-cells expressing VAMP1. Error bars represent standard deviation of four

VAMPs 1, 3, 4, 5, 7 and 8 are expressed at the same level at the cell surface, while

**t-SNARE Syntaxin1/SNAP-25**

plasma membrane t-SNAREs.

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VAMPs 1, 2, 3, 7 and 8 are known to drive vesicle fusion with the plasma membrane. The above data provide additional evidence that vesicles that carry either one of these five VAMPs are capable of fusing with the plasma membrane. Since these VAMPs are functionally redundant, they may compensate each other in loss of function studies (Borisovska et al., 2005).

To rule out the possibility that the baseline -galactosidase activity detected in the VAMP5 combinations (Fig. 5) is caused by residual membrane fusion activity of VAMP5, we perform the microscopic cell fusion assay, which analyzes individual cell fusion events and can detect rare fusion events. In multiple experiments, no cell fusion is observed in the microscopic assay when VAMP5 is combined with syntaxin1/SNAP-25 or syntaxin4/SNAP-25, whereas VAMP4 drives fusion efficiently with both t-SNARE complexes (Hasan et al., 2010) (data not shown). Together, the results using enzymatic and microscopic cell fusion assays suggest that VAMP5 is unable to mediate membrane fusion with the plasma membrane t-SNAREs.

VAMP5 is expressed in muscle cells, in which it is mainly associated with the plasma membrane as well as intracellular vesicles (Zeng et al., 1998; Zeng et al., 2003). It is still possible that VAMP5 forms fusogenic interactions with other plasma membrane t-SNAREs to mediate exocytosis in muscle cells. Indeed, SNAP-23, a ubiquitously expressed SNAP-25 homolog, is expressed in skeletal muscle (Bostrom et al., 2010). The role of VAMP5 in exocytosis needs further investigation.
