Cell-free in vitro assays offer several advantages for elucidating molecular mechanisms underlying various biological processes. Here, we describe a simple and quantitative in vitro assay using isolated yeast microsomes to measure homotypic ER membrane fusion. In this assay, membrane fusion between ER microsomes is monitored by reconstitution of luciferase activity from split luciferase fragments. Our findings reveal that homotypic ER membrane fusion requires not only Sey1p, the yeast atlstin, but also ER-resident SNAREs, such as Sec22p and Sec20p, in Saccharomyces cerevisiae.
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