Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes.
Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes.
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DOI：
10.1083/jcb.149.1.55
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发表时间：
2000-04-03
期刊：
影响因子：
--
通讯作者：
Barlowe C
中科院分区：
文献类型：
--
作者：
Cao X;Barlowe C
可溶性NSF附着蛋白受体(SNARE)蛋白对于酵母ER和高尔基体之间转运过程中的膜融合是必不可少的。亚细胞分离实验表明，ER/高尔基体蛋白Bos1p、Sec22p、Bet1p、Sed5p和Rab蛋白Ypt1p分布相似，但主要定位于高尔基体膜。所有这些诱捕蛋白都被有效地包装到COPII囊泡中，表明诱捕机制在内质网和高尔基体之间动态循环。在这种情况下，Ypt1p不能有效地包装成囊泡。为了确定哪些膜蛋白功能是必需的，温度敏感的等位基因BOS1、BET1、SED5、SLY1和YPT1被用来选择性地使囊泡或高尔基膜上的这些基因产物失活。BOS1、BET1、SED5、SLY1和YPT1在限制温度下阻止ER/高尔基体体外运输。带有Bet1p或Bos1p突变的囊泡抑制与野生型受体膜的融合，但含有这些突变的受体膜是完全功能的。相反，带有Sed5p、Sly1p或Ypt1p突变的囊泡是有功能的，而含有这些突变的受体膜则阻止融合。因此，这组SNARE蛋白对称地分布在囊泡和受体之间，但它们的功能不对称，因此囊泡上需要Bet1p和Bos1p，受体膜上需要Sed5p活性。我们认为，SNARE蛋白功能的不对称性是通过在这种融合事件中对Ypt1p GTP酶的不对称分布和需求来维持的。当跨膜锚定形式的Ypt1p被用来将该GTP酶限制在受体隔室时，耗尽Ypt1p的囊泡仍然具有融合的能力。
Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.
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发表时间：
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期刊：
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影响因子：
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DOI：
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发表时间：
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