Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52246, USA.
J Cell Biol. 2011 Jan 24;192(2):229-42. doi: 10.1083/jcb.201008121. Epub 2011 Jan 17.
ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.
ESCRTs（内体分选复合物必需的运输）结合并隔离泛素化的膜蛋白，并将它们引导进入多泡体（MVBs）。作为泛素（Ub）结合蛋白，ESCRTs 本身也会被泛素化。然而，目前尚不清楚这是否调节了它们功能的一个关键方面，还是它们与 Ub 系统相关的非特异性后果。我们研究了 ESCRTs 的泛素化是否是其将货物分拣到 MVB 腔中的能力所必需的。尽管我们发现 Rsp5 是负责 ESCRT-0 泛素化的主要 Ub 连接酶，但消除 Rsp5 或消除 ESCRT-0 内可泛素化的赖氨酸都不会影响 MVB 的分拣。此外，通过将去泛素化肽酶的催化结构域融合到 ESCRTs 上，我们可以阻断 ESCRT 的泛素化和经历 Rsp5 依赖性泛素化的蛋白质的分拣。然而，融合到单个 Ub 部分的蛋白质被有效地递送到 MVB 腔中，这强烈表明在没有 ESCRT 泛素化的情况下，单个 Ub 足以分拣 MVBs。
