MacDonald Ewan, Urbé Sylvie, Clague Michael J
Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3BX, UK.
Traffic. 2014 Aug;15(8):879-88. doi: 10.1111/tra.12180. Epub 2014 Jul 3.
The endosomal deubiquitylase USP8 has profound effects on endosomal morphology and organisation. Previous reports have proposed both positive (EGFR, MET) and negative roles in the down-regulation of receptors (Frizzled, Smoothened). Here we report an additional influence of USP8 on the retromer-dependent shuttling of ci-M6PR between the sorting endosome and biosynthetic pathway. Depletion of USP8 leads to a steady state redistribution of ci-M6PR from the Trans-Golgi Network (TGN) to endosomal compartments. Consequently we observe a defect in sorting of lysosomal enzymes, evidenced by increased levels of unprocessed Cathepsin D, which is secreted into the medium. The normal distribution of receptor can be restored by expression of siRNA-resistant USP8 but not by a catalytically inactive mutant or a truncated form, lacking a MIT domain required for endosomal localisation. We suggest that effects of USP8 depletion may reflect the loss of ESCRT-0 components which associate with retromer components Vps35 and SNX1, whilst failure to efficiently deliver lysosomal enzymes may also contribute to the observed block in receptor tyrosine kinase degradation.
内体去泛素化酶USP8对内体形态和组织具有深远影响。先前的报道表明，USP8在受体（卷曲蛋白、 smoothened）的下调中既具有正向作用（表皮生长因子受体、间质上皮转化因子），也具有负向作用。在此，我们报道了USP8对阳离子独立型甘露糖6-磷酸受体（ci-M6PR）在分拣内体和生物合成途径之间依赖回收体的穿梭运输的额外影响。USP8的缺失导致ci-M6PR从反式高尔基体网络（TGN）到内体区室的稳态重新分布。因此，我们观察到溶酶体酶分拣存在缺陷，这通过分泌到培养基中的未加工组织蛋白酶D水平升高得以证明。通过表达对小干扰RNA（siRNA）具有抗性的USP8可以恢复受体的正常分布，但通过催化失活的突变体或缺少内体定位所需MIT结构域的截短形式则无法恢复。我们认为，USP8缺失的影响可能反映了与回收体成分Vps35和分选连接蛋白1（SNX1）相关的转运所需内体分选复合物（ESCRT-0）成分的丧失，而未能有效递送溶酶体酶也可能导致观察到的受体酪氨酸激酶降解受阻。
