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

虽然泛素信号对质膜内吞作用（PM）和反式高尔基网络（TGN）和核内体/多泡体分选的重要性已经被假设，但对泛素化调节转运的蛋白质知之甚少。SKD1 （E235Q）是SKD1/Vps4B的一种atp酶缺陷形式，它从异常核内体（EQ室；哺乳动物E类vps室）中积累的泛素化蛋白中消除了泛素的再循环。我们利用这一点，结合亲和纯化和LC-MS/MS鉴定泛素化底物。发现了多种定位于PM的受体、转运体、细胞粘附和信号分子，其中大多数以泛素化依赖的方式积聚在EQ室中。此外，整体膜蛋白2B和nedd4相互作用蛋白2是哺乳动物细胞中第一个例子，它们在高尔基体/TGN和核内体之间的运输是通过泛素化调节的。有趣的是，转铁蛋白受体（TfR）的转运与其泛素化状态高度相关，因为游离泛素的消耗抑制了TfR的泛素化，并恢复了其在EQ区室中的积累。综上所述，所鉴定的蛋白是膜相关蛋白的最佳候选蛋白，其运输受泛素化调节。这些数据为我们确定泛素依赖蛋白分选的机制基础提供了进一步的步骤。

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.