ENZYMOLOGY OF GOLGI STACK FORMATION

我们已经重建了高尔基体的形成， 囊泡的平均直径为60-90 nm。 我们在细胞内发现了一种 含有120和66 kD mol. wt（p120/66）， 这是组装过程的非常早期阶段所需要的。 我们 结果表明，这些蛋白质可能参与解开束缚， 来自胞质锚的VGM。 未拴系或活动的VGM然后 通过NSF和SNAP依赖性过程融合，形成更大的囊泡， 约200-300 nm直径。 我们将正式测试p120/66的作用 将VGM组装成堆栈。 另外我们 建议识别p120/66相互作用伙伴。 存在下 一种类似NSF的ATP酶，称为p97，大囊泡融合， 膜被压平成盘状囊。 我们建议使用p97 可能与较大囊泡的融合后扁平化有关。 在 此外，我们已经开始分离外周高尔基体膜， 蛋白质，并找到一个馏分，经过三个柱层析步骤， 以控制堆叠形成活动。 我们将继续 分馏方案，以确定必要的成分。 细胞溶质 蛋白质组分中含有55和33 kD的多肽（p55/33）， GTP的存在通过IQ抑制高尔基体泡状形成。 我们建议 这些蛋白质可以将GTP交换到高尔基体膜上， 蛋白 因此，这些蛋白质的特征有望 揭示了高尔基体相关的GTP受体， 抑制了IQ的影响。 因此，我们的目标是揭示新颖的 深入了解高尔基体堆栈从VGM形成的机制， 高尔基体可能通过以下方式阻止其完全囊泡化的过程 利用GTP依赖性反应。

We have reconstituted the formation of Golgi stack formation from small vesicles of 60-90 nm average diameter. We have identified a cytosolic fraction containing two proteins of 120 and 66 kD mol. wt (p120/66), which is required for very early stages of the assembly process. Our results suggest that these proteins may be involved in untethering of VGMs from a cytosolic anchor. The untethered or mobilized VGMs then fuse by an NSF and SNAP dependent process to form larger vesicles of about 200-300 nm diameter. We will formally test the role of p120/66 in mobilizing VGMs for their assembly into stacks. In addition, we propose to identify p120/66 interacting partners. In the presence of an NSF like ATPase called p97, the large vesicles fuse and the resulting membranes are flattened into disc shaped cisternae. We suggest the p97 may be involved in post fusion flattening of the larger vesicles. In addition, we have started fractionating peripheral Golgi membrane proteins and find a fraction, after three column chromatographic steps to contain the stack formation activity. We will continue with this fractionation scheme to identify the essential components. A cytosolic protein fraction containing polypeptides of 55 and 33 kD (p55/33) in the presence of GTP inhibits Golgi vesiculation by IQ. We propose that these proteins may be exchanging GTP onto a Golgi membranes associated protein. The characterization of these proteins, therefore, promises to reveal the Golgi associated recipient of GTP, which as a result inhibits the effects of IQ. Our aims therefore promise to reveal novel insights into the mechanism by which Golgi stacks form from VGMs and the process by which the Golgi may prevent its complete vesiculation by utilizing a GTP dependent reaction.