Resolution of organelle docking and fusion kinetics in a cell-free assay.
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 activation in detergent lysates but does not alter the measured kinetics of docking or fusion. Thus, the lag between docking and ALP activation reflects a lag between docking and fusion. Many vacuoles in the population undergo multiple rounds of fusion; methods are presented for distinguishing the first round of fusion from ongoing rounds of fusion. A simple kinetic model distinguishes between two rates, the rate of fusion and the rate at which fusion competence is lost, and allows estimation of the number of rounds of fusion completed.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA. ajm@semilog.org
Alexey J
AJ
Merz
William T
WT
Wickner
D000469:Alkaline Phosphatase / Q000378:metabolism; D002474:Cell-Free System; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008561:Membrane Fusion; D015388:Organelles / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014617:Vacuoles / Q000502:physiology*; D015003:Yeasts / Q000166:cytology
D005656:Fungal Proteins; D011498:Protein Precursors; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases