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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Resolution of organelle docking and fusion kinetics in a cell-free assay.
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Resolution of organelle docking and fusion kinetics in a cell-free assay.

机译：无细胞测定中细胞器对接和融合动力学的解析。

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摘要

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.
机译：隔室混合的体外测定已成为细胞器对接和融合的生化解剖学中的关键工具。许多此类测定法是通过报告蛋白的酶促修饰来测量区室混合。酵母液泡的同型融合通过碱性磷酸酶原（pro-ALP）的蛋白水解成熟的偶联测定来测量。在对接结束时观察到动力学滞后，其特征是获得了对抗SNARE试剂的抗性，以及ALP成熟。因此，我们询问促ALP成熟所需的时间是否对所测融合信号增加了动力学滞后。 Prb1p促进ALP成熟； Prb1p的过量生产会加速去垢剂裂解物中的ALP活化，但不会改变对接或融合的动力学。因此，对接和ALP激活之间的延迟反映了对接和融合之间的延迟。种群中的许多液泡都经历了多轮融合。提出了区分第一轮融合与正在进行的融合的方法。一个简单的动力学模型可以区分两种速率，即融合速率和融合能力丧失的速率，并可以估算完成的融合次数。

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