Resolution of organelle docking and fusion kinetics in a cell-free assay

Abstract

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.Pho8p ͉ protease B ͉ SNARE ͉ Rab͞Ypt ͉ tethering M embrane docking and fusion events have been measured by many methods. Among the most commonly used are in vitro, coupled enzymatic assays of compartment mixing between two vesicle populations (1). One vesicle population contains a reporter substrate, and the other vesicle population contains an enzyme that can covalently modify the reporter substrate. We call vesicles containing the modifying enzyme ''effector'' vesicles and vesicles containing the substrate ''reporter'' vesicles. Fusion causes content mixing between effector and reporter vesicles, allowing the enzyme access to the reporter substrate. The amount of reporter modified thereby indicates the extent of fusion.Coupled transport and fusion assays have made possible the identification of molecules that mediate vesicle budding, docking and fusion, and the dissection of the mechanisms by which these molecules act. However, coupled assays have potential limitations. Treatments that inhibit the reporter system may be interpreted incorrectly as fusion inhibitors. Moreover, if the enzymatic processing of the reporter substrate is slow relative to the docking or fusion reaction of interest, the kinetics of the processing event will dominate, and thus obscure, the kinetics of fusion.We study yeast vacuole͞vacuole homotypic fusion. A key tool in our work is a cell-free, coupled enzymatic assay of vacuole fusion (2). In this assay, the reporter substrate is the enzyme alkaline phosphatase (ALP), encoded by the PHO8 gene. ALP is synthesized as an inactive precursor, pro-ALP, and is processed into an active form by proteolytic cleavage (3, 4). The reporter vacuoles contain pro-ALP but are protease deficient. The effector vacuoles are protease replete but lack pro-ALP. Content mixing between effector and reporter vacuo...

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

“…3 B-D) without triggering a substantial burst of fusion first. These findings were similar to observations with intact vacuoles, where docking occurs ∼20 min before fusion (12,27).…”

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

“…Addition of soluble recombinant SNARE Vam7p (22) permits synchronous SNARE pairing (15). The reaction rapidly acquires insensitivity to Vam3p antibody (half-time Ͻ1 min), whereas content mixing occurs relatively slowly, with a half-time of 15 min (20,21). Strikingly, after rVam7p addition the R18 dequenching (Fig.…”

“…This is however unlikely: (i) Although the vacuolar protease Prb1p is largely responsible for ALP maturation, Prb1p overproduction has no effect on the kinetics of fusion-dependent ALP maturation (20), showing that Prb1p had not been limiting to maturation. (ii) Mature ALP appears in vivo with a half-life of 6 min (24), which includes its synthesis and transport through the endoplasmic reticulum and Golgi to the vacuole; thus the proteolytic maturation of pro-ALP in the vacuole takes much less than 6 min.…”

“…Its liberation by Sec17p/18p/ATP allows trans-SNARE complex assembly. By incubating vacuoles with anti-Sec17p antibodies (20,21), tethering can occur, but trans-SNARE pairing is prevented by the absence of free Vam7p. Addition of soluble recombinant SNARE Vam7p (22) permits synchronous SNARE pairing (15).…”

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

“…Ypt7p and HOPS are needed for SNARE complex assembly, and HOPS is an integral part of the assembled SNARE complex (26). Fusion occurs around the vertex ring, joining the outside membrane from each vacuole to form the larger, fused organelle and joining the two apposed boundary membrane discs to yield a luminal vesicle (22,27).…”