The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles
The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles
复制标题
DOI：
10.1038/sj.emboj.7601486
复制
发表时间：
2007-01-24
期刊：
影响因子：
11.4
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
--
作者：
Kleijnen, Maurits F.;Kirkpatrick, Donald S.;Gygi, Steven P.
众所周知，泛素化在没有蛋白酶体参与的情况下调节细胞内囊泡运输的早期阶段。我们现在表明，在酵母中，泛素化调节后期，膜融合，与蛋白酶体参与。一个已知的蛋白酶体突变体在体内具有空泡断裂表型，通常与空泡膜融合缺陷相关，表明蛋白酶体在融合中起作用。抑制空泡蛋白酶体干扰了体外膜融合，表明没有蛋白酶体降解就不能发生融合。如果是这样，人们就会期望在液泡膜上发现泛素化蛋白。我们发现了其中的一小部分，确定了最普遍的一个是Ypt7，并绘制了它的两个主要泛素化位点。泛素化的Ypt7与融合所必需的降解事件有关：空泡型Ypt7和空泡型蛋白酶体相互依赖，泛素化的Ypt7在融合过程中成为蛋白酶体的底物，当我们降低Ypt7泛素化时，蛋白酶体抑制剂会更大程度地降低融合。最强的模型认为，没有泛素化的Ypt7的蛋白酶体降解，融合就不能进行。由于Ypt7是众多Rab gtpase之一，泛素蛋白酶体的调控可能参与了其他地方的膜融合。
Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin-proteasome regulation may be involved in membrane fusion elsewhere.