On and off membrane dynamics of the endoplasmic reticulum-Golgi tethering factor p115 in vivo
On and off membrane dynamics of the endoplasmic reticulum-Golgi tethering factor p115 in vivo
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DOI：
10.1091/mbc.e05-09-0862
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发表时间：
2006-07-01
影响因子：
3.3
通讯作者：
Sztul, Elizabeth
中科院分区：
文献类型：
--
作者：
Brandon, Elizabeth;Szul, Tomasz;Sztul, Elizabeth
在体内调节p115系聚因子的膜募集的机制尚不清楚。在这里，我们描述了p115在膜和细胞质之间的循环，并记录了高尔基基质蛋白、Rab1和可溶性n -乙基酰亚胺敏感因子（NSF）附着蛋白（SNAP）受体（SNAREs）在这一过程中的作用。快速的膜/细胞质交换表现为反复光漂白细胞外周后高尔基定位的p115-绿色荧光蛋白（GFP）迅速（t（1/2）类似于20秒）丧失，光漂白高尔基定位的p115-GFP后迅速（t（1/2）类似于13秒）荧光恢复。缺少GM130/giantin结合位点的p115突变体在光漂白后表现出类似的荧光恢复（FRAP） （t（1/2）类似于13 s），这表明GM130和giantin不是p115膜动力学的主要决定因素。相比之下，在表达失活Rab1/N121I突变体的细胞中，p115- gfp交换更快（t（1,2）与8 s相似），表明p115循环受到Rab1的影响。p115-GFP动力学也受到SNAREs装配状态的影响。在表达atpase缺陷的NSF/E329Q突变体抑制SNARE复合物分解的细胞中，p115-GFP的循环动力学明显减慢（t(1,2)，类似于21秒）。相比之下，在抑制囊泡交通的低温（10℃）下培养的细胞中，p115-GFP的循环动力学更快（t（1/2）类似于7秒）。这些数据表明膜上的p115结合位点是由未组装的SNAREs提供的。与此一致的是，生化研究表明，在NSF和α - snap存在的情况下，p115向膜的募集增加。我们的数据支持一个模型，在该模型中，系绳的招募直接受到SNAREs装配状态的调节。
The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t(1/2) similar to 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t(1/2) similar to 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t(1/2) similar to 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t(1,2) similar to 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t(1,2) similar to 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t(1/2) similar to 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.