Beck K A, Chang M, Brodsky F M, Keen J H
Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Cell Biol. 1992 Nov;119(4):787-96. doi: 10.1083/jcb.119.4.787.
We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.
我们研究了已去除表面包被的网格蛋白包被囊泡的体外行为，使得大部分网格蛋白被去除，但仍有大量网格蛋白组装蛋白（AP）与膜结合。当将这些去除包被的囊泡（s-CV）置于接近细胞内pH和离子强度（pH 7.2，约100 mM盐）的条件下时，可观察到它们的聚集。这种s-CV聚集反应迅速（t1/2≤0.5分钟），在4-37℃范围内与温度无关，且不受ATP、鸟苷-5'-O-（3-硫代磷酸），特别是EGTA的影响，这使其与依赖Ca(2+)的膜聚集反应区分开来。该过程由与膜结合的AP分子的作用驱动，因为部分蛋白水解会导致活性完全丧失，并且用与AP-2的α亚基反应的单克隆抗体对s-CV进行预处理可消除聚集。然而，囊泡聚集不受焦磷酸（PPPi）抑制，表明先前表征的对多磷酸盐敏感的AP-2自缔合与该反应无关。囊泡聚集反应可以重建：磷脂组成接近质膜和包被囊泡细胞质表面上发现的脂质体（70% L-α-磷脂酰乙醇胺（I-A型）、15% L-α-磷脂酰-L-丝氨酸和15% L-α-磷脂酰肌醇）在添加AP-2后聚集，但添加AP-1、AP-3（AP180）或纯网格蛋白三脚复合体后不聚集。用胰蛋白酶对AP-2进行有限的蛋白水解可消除脂质体的聚集。此外，一种高度纯化的不含β的AP-2α制剂会导致脂质体聚集，而纯β亚基则不会，这与在s-CV测定中获得的结果一致，该结果也表明α亚基参与其中。使用荧光能量转移测定法，我们表明在生理溶液条件下AP-2不会导致脂质体融合。然而，由于膜融合必然需要两个参与的双层紧密相对，我们所确定的依赖AP-2的囊泡聚集事件可能代表体内内吞作用和网格蛋白去包被后发生的内体形成和融合的初始步骤。
