Orci L, Malhotra V, Amherdt M, Serafini T, Rothman J E
Department of Morphology, University of Geneva Medical School, Switzerland.
Cell. 1989 Feb 10;56(3):357-68. doi: 10.1016/0092-8674(89)90239-0.
We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.
我们利用一种无细胞系统，该系统基本上重构了一轮水泡性口炎病毒（VSV）编码的G蛋白在高尔基体潴泡之间的运输过程，以识别载体囊泡成熟过程中的离散阶段。使用GTPγS和N - 乙基马来酰亚胺（NEM）分别作为选择性抑制剂来积累有被小泡和无被小泡，我们发现它们是连续且必不可少的运输中间体。我们发现，当运输受阻时积累的有被小泡和无被小泡已经从供体潴泡转移到受体潴泡，但尚未融合。类似的有被小泡和无被小泡也会在经过适当处理的完整细胞中积累。我们的研究表明，一种类似于负责受体介导的内吞作用的有被芽（小窝）-有被小泡-无被小泡系统介导生物合成蛋白跨高尔基体潴泡的运输。然而，“高尔基体”有被芽不含网格蛋白，似乎作为批量载体起作用，而内吞性网格蛋白包被小窝携带高度选择性的货物。
