Ren Jihui, Pashkova Natasha, Winistorfer Stanley, Piper Robert C
Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
J Biol Chem. 2008 Aug 1;283(31):21599-611. doi: 10.1074/jbc.M802982200. Epub 2008 May 28.
Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.
泛素（Ub）是一种分选信号，它通过将整合膜蛋白引导至多泡体（MVB）的腔内小泡，使其靶向液泡/溶酶体内部。Vps27-Hse1复合物与哺乳动物细胞中的Hrs-STAM复合物同源，在早期内体表面作为Ub分选受体发挥作用。我们发现Hse1与Doa1/Ufd3相互作用。已知Doa1与Cdc48/p97和Ub相互作用，并且是维持Ub水平所必需的。我们发现Hse1的Src同源3结构域直接与Doa1的中央PFU结构域结合。Doa1中阻止Hse1结合但不阻止Ub结合的突变不会改变Ub水平，但会导致MVB货物GFP-Cps1的分选错误。与Vps27缺失相结合时，Doa1的缺失也会导致合成生长缺陷。与单独缺失Doa1不同，doa1Delta vps27Delta双突变体表型不会被Ub过表达所抑制，这表明该效应不是由于Ub水平降低的间接后果。Doa1的缺失导致酵母液泡内GFP-Ub积累缺陷，这意味着通过MVB途径的泛素化膜蛋白通量减少。这种缺陷也反映在无法正确分选Vph1-GFP-Ub上，Vph1-GFP-Ub是多蛋白液泡ATP酶复合物的一个修饰亚基，它携带一个作为MVB分选信号的Ub框内融合蛋白。这些结果揭示了Doa1在帮助处理泛素化膜蛋白以便分选到MVB中的新作用。
