Traffic and expression of membrane proteins regulated by an ubiquitin-lysosome system

尽管泛素信号对细胞膜(PM)的内吞作用、跨高尔基体(TGN)和内体/多囊泡小体的分选都有重要意义，但其转运受泛素化调控的蛋白质却知之甚少。SKD1(E235Q)是一种ATPase缺陷形式的SKD1/Vps4B，它取消了泛素从异常内体(EQ隔室；哺乳动物E类VPs隔室)中积累的泛素化蛋白中的循环。我们利用这一点利用亲和纯化和LC-MS/MS相结合的方法来鉴定泛素化底物。我们鉴定了定位于PM的各种受体、转运体、细胞黏附和信号分子，其中大多数以泛素化依赖的方式聚集在EQ室中。此外，完整的膜蛋白2B和Nedd4相互作用蛋白2是哺乳动物细胞中的第一个例子，其在高尔基体/TGN和内体之间的运输受到泛素化的调节。有趣的是，转铁蛋白受体(TFR)的运输与其泛素化状态高度相关，因为游离泛素的耗竭抑制了TFR的泛素化，并恢复了它在EQ间隔室的积聚。综上所述，所鉴定的蛋白质是膜相关蛋白的最佳候选者，其转运受泛素化调控。这些数据为我们确定泛素依赖的蛋白质分类的机制基础提供了前进的步骤。

Although the significance of ubiquitin-signals for both endocytosis from the plasma membrane (PM) and sorting at trans-Golgi network (TGN) and endosomes/multivesicular body has been postulated, the proteins whose trafficking is regulated by ubiquitylation are poorly understood. SKD1 (E235Q), an ATPase-deficient form of SKD1/Vps4B, abrogates the recycling of ubiquitin from the ubiquitylated proteins accumulated in aberrant endosomes(EQ compartment; mammalian class E vps compartment). We have taken advantage of this to identify substrates for ubiquitylation using a combination of affinity purification and LC-MS/MS. A variety of receptors, transporters, cell adhesion and signaling molecules localized to the PM are identified, and most of them are accumulated in EQ compartment in an ubiquitylation-dependent manner. Moreover, integral membrane protein 2B and Nedd4-interacting protein 2 are the first examples in mammalian cells, of proteins whose trafficking between the Golgi/TGN and endosomes is regulated by ubiquitylation. Intriguingly trafficking of transferrin receptor (TfR) is highly correlated with its ubiquitylation state, since free ubiquitin depletion inhibits ubiquitylation of TfR and resumes its accumulation in EQ compartments. Taken together, the identified proteins are the best candidates for the membrane-associated proteins whose trafficking is regulated by ubiquitylation. These data provide us forward steps to identify the mechanistic basis for ubiquitin-dependent protein sorting.