Robinson M S, Pearse B M
J Cell Biol. 1986 Jan;102(1):48-54. doi: 10.1083/jcb.102.1.48.
A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.
一类包被囊泡蛋白，分子量约为100,000，命名为100K，与包被组装以及网格蛋白与囊泡膜的附着都有关联。这些蛋白质通过凝胶过滤、羟基磷灰石层析和制备性SDS-PAGE从牛脑包被囊泡提取物中纯化得到。通过有限蛋白酶解进行肽图谱分析表明，构成三个主要100K条带的多肽具有不同的氨基酸序列。当用总100K蛋白免疫四只大鼠时，每只大鼠对不同条带的反应不同，尽管所有四种抗血清都与人胎盘包被囊泡的100K蛋白发生交叉反应。亲和纯化后，其中两种抗血清能够在3T3全细胞蛋白印迹上检测到100K条带，并用于免疫荧光，用兔抗网格蛋白或小麦胚凝集素作为高尔基体标记对细胞进行双重标记。两种抗血清给出的染色都与抗网格蛋白一致，在质膜上有斑点状标记，在核周高尔基体上也有标记。因此，100K蛋白存在于内吞作用以及源自高尔基体的包被小窝和囊泡上。抗100K和抗网格蛋白的斑点模式几乎相同，表明当囊泡脱包被时，100K蛋白和网格蛋白一起被去除。与抗网格蛋白抗血清相比，两种抗血清中的一种在质膜上的标记比在高尔基体上的标记更强。另一种抗血清在高尔基体上的标记更强。尽管我们目前还没有证据表明这两种抗血清在3T3细胞印迹上标记不同的蛋白质，但它们在牛脑100K蛋白印迹上确实显示出差异。这个结果虽然是初步的，但增加了不同的100K蛋白可能与不同的膜运输途径相关的可能性。
