Institut Jacques Monod, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7592, Université Paris-Diderot, Sorbonne Paris Cité, Paris, France.
Mol Biol Cell. 2012 Jun;23(11):2170-83. doi: 10.1091/mbc.E11-10-0891. Epub 2012 Apr 4.
In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.
在酵母中，跨膜蛋白被分拣到多泡体（MVB）内部囊泡中需要其被泛素连接酶 Rsp5 泛素化。这使得它们能够被几种内体分选复合物必需的运输（ESCRT）亚基的泛素结合域（UBD）识别。K63 连接的泛素（K63Ub）链修饰了几种 MVB 货物，因此我们表明它们主要定位于 E 类隔室，该隔室在缺乏 ESCRT 成分的细胞中积累泛素化的货物。相反，无法生成 K63Ub 链的酵母细胞表现出 MVB 分拣缺陷。这些特性在真核生物中是保守的，因为哺乳动物黑素体 MVB 货物 MART-1 被 K63Ub 链修饰，并且当这些链的生成受到抑制时部分错误分拣。我们表明，所有含有 UBD 的酵母 ESCRT 蛋白都经历泛素化和去泛素化，其中一些通过 Rsp5 和泛素异肽酶 Ubp2 的相反活性进行修饰，已知这两种酶分别优先组装和解组装 K63Ub 链。酵母中无法生成 K63Ub 链会导致 MVB 超微结构改变。因此，我们的工作揭示了 K63Ub 链在货物分拣和 MVB 发生中的双重功能。
