**7. References**

224 Crosstalk and Integration of Membrane Trafficking Pathways

respectively. As internal standards of the cDNA amounts, GAPdH fragments were amplified with primers 5'-CCGGACAAGGCTGCTGCTAC-3' (GAP FW) and 5'- GACCCTCCACAATGCCAAACC-3' (GAP RW), designed on the basis of the tobacco GAPdH (cytosolic glyceraldehyde-3-phosphate dehydrogenase) gene (Accession number:

The coding region of *NtNRAMP1* was amplified by PCR using gene specific primers of 5'- CACCATGGCGGCGAACTCGTCCCC-3' and 5'-ATTAGTGGTCCTCTGCTGAGGCAA-3', then cloned into the pENTR/D-TOPO vector (Invitrogen) and finally introduced into the pGWB502 binary vector (Nakagawa et al. 2007) by the Gateway cloning system using LR clonase (Invitrogen). The pGWB502 vector gave the cauliflower 35S promoter sequence to the PCR products. *Agrobacterium*-mediated transformation of the tobacco BY-2 cells was performed as described by Mayo et al. (2006). Transformants were selected with 50 mg l-1

Transient gene expression was carried out by particle bombardment. A cell suspension of 2 d-old BY-2 cells was filtrated onto filter paper, and the cells bombarded with gold particles (1.0 m) coated with the appropriate vector constructs using a particle delivery system (PDS-1000/He, Bio-Rad, Hercules, CA, USA) according to the manufacturer's recommendations. Filtrated BY-2 cells were placed at a distance of 6 cm under the stopping screen and were bombarded in a vacuum of 28 inches Hg at a helium pressure of 1100 psi. Following bombardment, the cells were diluted in LSD medium and kept in the dark at 27 C for 6 to 12 h before observation. The GFP fluorescence was detected on the inverted platform of a fluorescence microscope equipped with a spinning disc confocal laser scanning system (CSU-X1, Yokogawa, Tokyo, Japan) and a cooled CCD camera (Cool-SNAP

Yeast cells INVSc1 (Invtrogen) were transformed by pYES2.1/V5-His-TOPO vectors (Invitrogen) containing an entire ORF region of the respective metal transporter cDNAs according to standard procedures (Invitrogen). The transformants were selected on synthesic complete medium omitted uracil (SC-uracil) containing 2 % glucose, 0.67 % yeast nitrogen base (without amino acids, Difco), amino acids omitting uracil (-Ura DO Supplement, Clontech Laboratories Inc.), 0.5 % ammmoniumu sulfate and 2 % agar. The transporter proteins were induced by application of 2 % galactose instead of glucose in the SC-uracil medium. For iron uptake measurements, yeast cells precultured in the SC-uracil medium were diluted to OD600 of 0.3 and cultured in the medium supplied with 2 % galactose and 0.2 mM FeCl3. After 18 h incubation, OD600 were measured and the yeast culture was washed with deionized water twice. For iron extraction, yeast cells were digested with 2N HCl and the iron concentrations were determined by atomic absorption

We are grateful to Dr. T. Nakagawa (Shimane University) for the kind gift of the pGWB502

M14419, Dambrauskas et al., 2003) and the relative transcript values then calculated.

**5.6 Transformation of tobacco BY-2 cells** 

HQ, PhotoMetrics, Huntington Beach, Canada).

spectrograph (AA-660, Shimadzu Co., Kyoto, Japan).

**5.7 Yeast experiments** 

**6. Acknowledgment** 

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hygromycin.


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**11** 

*USA* 

**Analysis of SNARE-Mediated** 

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

> **Vesicle fusion**

**Exocytosis Using a Cell Fusion Assay** 

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).

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

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-

**SNARE complex formation**

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

**1. Introduction** 

**Plasma membrane**

**Transport vesicle**

**t-SNARE**

**v-SNARE (VAMP)**

