title: Analysis of SNARE-mediated membrane fusion using an enzymatic cell fusion assay.
abstract: The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion(1-4). Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion(5). In a cell fusion assay(6,7), SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively. Here we describe a new assay(8) that quantifies SNARE-mediated cell fusion events by activated expression of β-galactosidase. Two components of the Tet-Off gene expression system(9) are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of β-galactosidase. The activity of β-galactosidase is quantified using a colorimetric method by absorbance at 420 nm. The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments(10-15). By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, KY, USA.
authors.forename: Nazarul
authors.initials: N
authors.lastname: Hasan
authors.forename: David
authors.initials: D
authors.lastname: Humphrey
authors.forename: Krista
authors.initials: K
authors.lastname: Riggs
authors.forename: Chuan
authors.initials: C
authors.lastname: Hu
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D008561:Membrane Fusion / Q000502:physiology*; D008856:Microscopy, Fluorescence; D050600:SNARE Proteins / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D050600:SNARE Proteins; D001616:beta-Galactosidase