Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
Nat Protoc. 2010 Apr;5(4):758-72. doi: 10.1038/nprot.2010.38. Epub 2010 Apr 1.
The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defined Golgi disassembly and reassembly assay that reconstitutes this process using purified proteins instead of cytosol. Treatment of Golgi membranes with mitotic kinases and COPI coat proteins efficiently disassembles the membranes into mitotic Golgi fragments, whereas further incubation with p97 or N-ethylmaleimide-sensitive factor (two AAA ATPases involved in membrane fusion) and their cofactors, in combination with protein phosphatase PP2A, leads to reassembly of the membranes into new Golgi stacks. The whole process takes 3-4 d and is applicable for identification and determination of novel cytosolic and membrane proteins that regulate Golgi membrane dynamics in the cell cycle.
高尔基器在有丝分裂期间经历广泛的解体，并在有丝分裂后的子细胞中重新组装。通过用有丝分裂和间期间质处理高尔基膜，在体外模拟了这个过程。为了确定控制形态变化的最小机制，我们开发了一种定义明确的高尔基解体和再组装测定法，该方法使用纯化的蛋白质而不是细胞质来重新构建这个过程。用有丝分裂激酶和 COPI 衣壳蛋白处理高尔基膜，可有效地将膜分解成有丝分裂的高尔基片段，而进一步与 p97 或 N-乙基马来酰亚胺敏感因子（两种参与膜融合的 AAA ATP 酶）及其辅助因子孵育，并与蛋白磷酸酶 PP2A 结合，导致膜重新组装成新的高尔基堆栈。整个过程需要 3-4 天，可用于鉴定和确定新型细胞质和膜蛋白，这些蛋白调节细胞周期中的高尔基膜动力学。
