Data from: Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 second lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

ESCRT复合物（ESCRT machinery）介导逆向膜剪切过程。通过定量荧光点阵光片显微镜（quantitative fluorescence lattice light-sheet microscopy）技术，我们证实ESCRT-III亚基可在酵母内体上快速聚合，并伴随至少两个Vps4六聚体的招募。在其3~45秒的存续周期内，ESCRT-III组装体可累积75~200个Snf7分子与15~50个Vps24分子。功能性出芽事件至少需要额外招募两个Vps4六聚体。膜出芽过程与Vps4和ESCRT-III组分的持续随机交换相关，而非固定组装体的稳态增长，且该过程依赖Vps4的ATP酶活性。一个全有或全无的步骤最终介导了ESCRT-III与Vps4的释放。断层电子显微镜（tomographic electron microscopy）实验证实，急性阻断Vps4的招募会阻滞膜出芽过程。我们提出如下模型：多个Vps4六聚体（四个及以上）可将多条ESCRT-III丝束聚集在一起。该过程会引发货物拥挤与膜向内凹陷，随后使新生出芽颈收缩，最终通过囊泡分裂产生腔内囊泡（ILV, intralumenal vesicles）。