SNARE Function Is Not Involved in Early Endosome Docking
SNARE Function Is Not Involved in Early Endosome Docking
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DOI：
10.1091/mbc.e08-05-0457
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发表时间：
2008-12-01
影响因子：
3.3
通讯作者：
Rizzoli, Silvio O.
中科院分区：
文献类型：
--
作者：
Geumann, Ulf;Barysch, Sina Victoria;Rizzoli, Silvio O.
转运囊泡的对接和融合构成细胞内膜运输的基本步骤。虽然对接被认为是由Rab-effector复合物启动的，但融合是由SNARE（N-乙基马来酰亚胺敏感因子[NSF]附着受体）蛋白介导的。然而，最近一直在争论SNARE是否也在建立或维持稳定停靠状态中发挥作用。为了解决这个问题，我们研究了SNARE依赖的对接和融合的早期内体，中央分选室的内吞途径之一。开发了一种新的基于荧光的体外试验，这使我们能够并行研究融合和对接。与同型融合类似，早期内体的对接依赖于ATP的存在并且需要生理温度。与融合不同，对接对可溶性SNARE基序、SNARE特异性F-ab片段或NSF活性阻断引起的SNARE功能扰动不敏感。相反，正如预期的那样，通过干扰磷脂酰肌醇（PI）-3磷酸的合成、Rab-GTP酶的功能以及早期内体自身抗原1（EEA 1）（一种必需的拴系因子），对接强烈减少。我们的结论是，对接的早期内涵体是独立的SNARE功能。
Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F-ab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.