MECHANISMS OF GOLGI VESICULATION DURING MITOSIS

我们重建了高尔基体膜的碎裂和分散 体外培养通透性正常大鼠肾细胞 有丝分裂胞浆和32℃下的ATP再生系统 有丝分裂胞浆耗尽p34cdc2激酶(这是 进入有丝分裂)会导致高尔基体碎裂。耗尽或 胞浆有丝分裂原激活的蛋白激酶失活 然而，(MEK1)抑制高尔基体碎裂。将MEK1添加回MEK1 耗尽的有丝分裂胞浆可恢复高尔基体碎裂。我们发现， 下游(和相关的)胞浆MAPK、ERK1和ERK2不是 此过程所需的。有趣的是，一种MAPK，由 与ERK2具有广泛交叉反应的抗体密切相关 有高尔基膜。我们对这项提议的总体目标是 了解MEK1的激活机制并测试我们的 高尔基体丝裂原蛋白激酶直接下游靶点的假设 激活了MEK1。我们的研究应该会揭示监管者的身份 称为MEK1-激活MEK1所必需的激酶，其成分是 参与了碎裂的高尔基膜的分散，最后 高尔基膜相关蛋白，包括MEK1底物 所需的高尔基碎裂。这些研究应该提供一个低于- 高尔基体膜广泛存在的机制 有丝分裂开始时的碎裂。

We have reconstituted the fragmentation and dispersal of Golgi membranes by incubation of permeabilized Normal Rat Kidney (NRK) cells with mitotic cytosol and an ATP regenerating system at 32 C. We find that mitotic cytosol depleted of p34cdc2 kinase (the kinase necessary for entry into mitosis) causes Golgi fragmentation. Depletion or inactivation of the cytosolic Mitogen activated protein kinase kinase (MEK1), however, inhibits Golgi fragmentation. Adding back MEK1 to MEK1 depleted mitotic cytosol restores Golgi fragmentation. We find that the downstream (and the relevant) cytosolic MAPkinase, ERK1 and ERK2 are not required for this process. Interestingly, a MAPkinase, recognized by an antibody with broad cross-reactivity to ERK2 is tightly associated with Golgi membranes. Our overall goals for this proposal are to understand the mechanism by which MEK1 is activated and test our hypothesis that Golgi MAPkinase is the immediate downstream target of activated MEK1. Our studies should reveal the identity of a regulator termed MEK1-kinase necessary for MEK1 activation, components that are involved in dispersal of the fragmented Golgi membranes and finally the Golgi membrane associated proteins, including the MEK1 substrate required Golgi fragmentation. These studies should provide an under- standing of the mechanism by which Golgi membranes undergo extensive fragmentation at the onset of mitosis.