Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.
Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.
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DOI：
10.1083/jcb.139.5.1169
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Burgess DR
中科院分区：
文献类型：
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作者：
Fath KR;Trimbur GM;Burgess DR
细胞质动力蛋白是一种微管负端定向运动，被认为是极化上皮细胞中将囊泡从TGN运送到顶叶皮质的动力。从原代极化的肠上皮细胞分离的反式高尔基体富集膜含有基于肌动蛋白的肌球蛋白-I和动力蛋白，而分离的高尔基体堆叠没有动力蛋白但含有肌球蛋白-I(Fath，K.R.，G.M.Trimbur和D.R.Burgess)。1994年。J.细胞生物学。126：661-675)。我们现在证明，高尔基体在没有ATP的情况下，在体外与胞浆提供的动力蛋白结合，当与胞浆和ATP孵育时，芽形成小的膜。胞液中的动力蛋白结合到堆叠的区域，这些区域注定要发芽，因为动力蛋白存在于萌发的膜中，但在萌发后没有出现在堆叠中。在体外运动性分析中，萌发的膜只向微管末端移动。提取研究表明，动力蛋白与一种高尔基体外周膜蛋白(S)结合，该蛋白抵抗冰冷Triton X-100的提取。在胞浆存在的情况下，这些膜上的幽灵可以向微管的负端移动。洗涤剂提取的高尔基体和含有TGN的膜与由部分血影蛋白和锚蛋白组成的无定形基质密切相关。尽管已提出将动力蛋白与细胞器膜连接起来，但我们发现功能性动力蛋白可以独立于动力蛋白和锚蛋白与提取膜结合。
Cytoplasmic dynein is a microtubule minus-end–directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661–675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.