Zick Michael, Wickner William T
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, United States.
Elife. 2014 Sep 25;3:e03251. doi: 10.7554/eLife.03251.
Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.
过去使用重组蛋白脂质体进行的实验，采用从荧光脂质去淬灭推断膜融合的检测方法，表明仅液泡SNARE就足以在体外催化膜融合。虽然我们能够重复这些结果，但在更严格的内腔区室混合检测中，我们检测到的融合非常少。探索脂质去淬灭和内容物混合检测之间的差异时，我们惊讶地发现荧光脂质相对于SNARE的分布有显著影响。在没有其他蛋白质的情况下，反式SNARE的缔合会导致脂质去淬灭，这不能归因于融合或半融合。有效的内腔区室混合需要携带SNARE的蛋白脂质体的拴系。虽然生理性HOPS拴系复合物使反式SNARE缔合增加了几倍，但内容物混合速率增加了100多倍。因此，拴系在促进膜融合中发挥作用，其作用不仅仅是简单地增加总反式SNARE复合物的量。
