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Resolution Of Organelle Docking And Fusion Kinetics In A Cell-Free Assay, Alexey J. Merz, William T. Wickner
Resolution Of Organelle Docking And Fusion Kinetics In A Cell-Free Assay, Alexey J. Merz, William T. Wickner
Dartmouth Scholarship
In vitro assays of compartment mixing have been key tools in the biochemical dissection of organelle docking and fusion. Many such assays measure compartment mixing through the enzymatic modification of reporter proteins. Homotypic fusion of yeast vacuoles is measured with a coupled assay of proteolytic maturation of pro-alkaline phosphatase (pro-ALP). A kinetic lag is observed between the end of docking, marked by the acquisition of resistance to anti-SNARE reagents, and ALP maturation. We therefore asked whether the time taken for pro-ALP maturation adds a kinetic lag to the measured fusion signal. Prb1p promotes ALP maturation; overproduction of Prb1p accelerates ALP …