Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.
Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.
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DOI：
10.1083/jcb.139.5.1183
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Gruenberg J
中科院分区：
文献类型：
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作者：
Gu F;Aniento F;Parton RG;Gruenberg J
在本文中，我们证明了在限制性温度下，尽管内化和再循环仍在继续，但在ε辅酶蛋白(εCOP)存在ts-缺陷的突变CHO细胞系(LDLF)中，从早期到晚期的内吞体转运受到抑制。早期的内小体看起来就像一簇簇的细管，没有典型的多囊泡区域，这些区域通常注定要在运输到晚期内小体的过程中成为囊泡中间产物。我们还发现，在限制性温度下孵育的LDLF细胞制备的胞浆中，来自BHK供体内体的这些囊泡的体外形成被抑制。尽管在限制性温度下，εCOP在LDLF细胞中迅速降解，但其他COP-I亚基的细胞量不受影响。尽管没有εCOP，我们仍然发现β，β‘和ζCOP的一个亚复合体在体外仍然被募集到BHK内体上，并且这种结合表现出内体COS的特征，即对于εS的刺激和对内体pH的敏感性。以往的研究表明，γ和δCOP不存在于内体上。然而，当αCOP缺失时，通常存在于内体上的εCOP不再被招募。相反，所有的COP亚基，除了明显的εCOP本身，在体外仍然以不依赖于pH的方式与BHK生物合成膜结合。因此，我们的观察表明，多囊泡内小体的生物发生与早期的内小体组织相耦合，并依赖于COP-I蛋白。我们的数据还表明，内体COP的膜结合和功能是可以剖析的：虽然β，β‘和ζCOP保留了结合内体膜的能力，但COP在运输中的功能似乎依赖于α和/或εCOP的存在。
In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP.