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.

Fig. 2.

Apparent kinetics of fusion are not changed by Prb1p overproduction in effector vacuoles. Fusion or detergent lysate (Insets) reactions contained reporter vacuoles obtained from strain BJ3505 and effector vacuoles from either strain AMY45 (control strain, circles) or AMY47 (Prb1p overproducer, triangles). (Insets) Graphs show the kinetics of ALP processing in dilute detergent lysates, with the same vacuole preparations used for the fusion experiments in the main graphs. (A) Kinetics of inhibition experiment. Master reactions were incubated at 27°C. At the intervals shown, 30-μl aliquots were removed from the master reactions and placed on ice (open symbols) or mixed with anti-Vam3p antibody and incubated at 27°C for the remainder of the 80 min (filled symbols). (B) Kinetics after synchronous docking. Master reactions were initiated as in A but contained anti-Sec17p IgG added from the start. After incubation for 20 min at 27°C, recombinant Vam7p (5 μM) was added to the master reactions, and at intervals, 30-μl aliquots were removed from the master reactions and placed on ice (open symbols) or mixed with anti-Vam3p antibody and incubated at 27°C for the remainder of the 80 min (filled symbols). The 0 time point in this experiment is the signal obtained with a reaction not rescued by Vam7p addition and was equivalent to the signal given in a reaction in which anti-Vam3p was added immediately before Vam7p. The ALP values are plotted in normalized form; maximum ALP activity values at 80 min were 3.6 units for the reactions containing control effector vacuoles (circles) and 4.5 units for the reactions containing Prb1p overproducer vacuoles (triangles).

Fig. 4.

Quantitative analyses of single and multiple rounds of fusion in the homotypic vacuole fusion system. (A) Cartoon depicting effects of initiating fusion reactions with different proportions of effector and reporter vacuoles. (B) Modeling of the quantitative consequences of varying effector and reporter vacuole fractions. Details of the model are given in Materials and Methods. For these simulations, the fusion rate constant was set at 0.03 min^–1. In both panels, the darker traces indicate reactions containing equal fractions of effector and reporter vacuoles, the standard reaction condition. (Left) The fraction of total vacuoles that are effectors. Because any vacuole that contains proteases is an effector, content mixing between effectors and reporters causes the fraction of effectors to increase over time. When reactions are initiated with large fractions of effectors, reporter vacuoles will, when they fuse, almost always fuse with an effector, yielding an ALP signal (Right). Under these conditions, only the first round of fusion contributes substantially to the ALP signal. At lower effector fractions, the fraction of effectors increases dramatically over time (Left), with the consequence that the ALP signal (Right) increases most rapidly during later fusion events. (C) Raw data from an experiment in which the initial effector and reporter were varied over a wide range. Background (ice incubation) values are subtracted from all signals; these values were 0.16, 0.27, and 1.0 units for reactions containing initial effector vacuole fractions of 0.025, 0.5, and 0.975, respectively. (D) Modeling as in B but with the incorporation of a rate constant for irreversible decay of fusion competence. The downward-sloping trace (Left) shows the decrease in proportion of fusion-competent vacuoles over time. For these simulations, the fusion rate constant k₁ was set at 0.03 min^–1, and the rate constant for loss of fusion competence k₂ was set at 0.01 min^–1.(E) Fits of the model described in D to the experimental data shown in C. Details are given in Materials and Methods.

Fig. 1.

Inhibitor sensitivity of in vitro ALP activation. (A) Fusion-dependent ALP activation. (B) ALP activation in dilute detergent lysates. Standard reactions were initiated as described in Materials and Methods. Inhibitors were added either at the start or the end of a 60-min incubation at 27°C. Inhibitor concentrations were 25 μM for IB2, 1 mM for PMSF, 50 μM for pepstatin, and 110 mM for anti-Vam3p.

Fig. 3.

Formation of large vacuoles as a consequence of in vitro fusion. (A and B) Standard fusion reactions were initiated in the absence or presence of anti-Vam3p antibody, as indicated, incubated for 120 min at 27°C, stained with MDY-64 fluorophore, and mounted for microscopy. (C) Vacuole diameters were measured from micrographs, and surface areas were estimated and plotted as histograms.