In vitro reconstitution of microtubule plus end-directed, GTPγS-sensitive motility of Golgi membranes
In vitro reconstitution of microtubule plus end-directed, GTPγS-sensitive motility of Golgi membranes
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DOI：
10.1091/mbc.9.10.2699
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发表时间：
1998-10-01
影响因子：
3.3
通讯作者：
Bloom, GS
中科院分区：
文献类型：
--
作者：
Fullerton, AT;Bau, MY;Bloom, GS
将纯化的高尔基体膜与细胞质和微管（MT）混合，并通过视频增强光学显微镜观察。最初，膜表现为沿着沿着MT移动的囊泡。随着时间的推移，囊泡形成聚集体，从中出现膜小管，沿着沿着MT行进，并最终产生广泛的网状网络。膜运动需要ATP，主要发生在MT+末端，并且几乎完全被驱动蛋白重链的H1单克隆抗体、5 '-腺苷酰亚胺二磷酸和100 μ M但不是20 μ M的钒酸盐抑制。运动也被GTP γ S或AlF 4-阻断，但对AlCl 3、NaF、星形孢菌素或冈田酸不敏感。GTP γ S和AlF 4-的目标显然是胞质起源，不包括驱动蛋白或MT，并且对三聚体G蛋白的几种探针不敏感。高尔基体膜沿着MT的运输由驱动蛋白介导，因此在体外重建。运动性由一种或多种胞质GTP酶调节，但不受分别被星形孢菌素或冈田酸抑制的蛋白激酶或磷酸酶的调节。相关的GTP酶可能是小G蛋白或发动蛋白。体外运动性可能对应于体内高尔基体至ER或高尔基体至细胞表面转运。
Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5'-adenylylimidodiphosphate, and 100 mu M but not 20 mu M vanadate. Motility was also blocked by GTP gamma S or AlF4- but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTP gamma S and AlF4- were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.