Abstract：
The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause the denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for the maintenance of PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes the mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p, the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.
摘要：
质膜（PM）不断暴露于来自细胞外环境的各种应力，如热和氧化应激。这些应力通常会导致膜蛋白变性并破坏PM完整性，这对正常的细胞活力和功能至关重要。为了维护PM完整性，大多数真核细胞具有PM质量控制（PMQC）系统，通过胞吞作用去除受损的膜蛋白。通过泛素介导的内吞作用从PM中去除受损的蛋白质是维持PM完整性的关键机制。但是早期核内体在PMQC系统中的重要性仍然没有得到很好的理解。在这里，我们展示了早期/分选内体功能中的关键蛋白质，Vps21p（酵母Rab5），Vps15p（磷脂酰肌醇-3激酶亚基），和Vps3p/8p（CORVET复杂亚基），参与维护PM的完整性。我们发现富含Vps21p的内体响应于热应力而改变PM附近的定位，然后迅速融合并形成扩大的隔室，以有效地将Can1p运输到液泡。此外,我们表明去泛素化酶Doa4p也参与PM的完整性，它的缺失导致Vps21p在液泡腔中的错误定位。有趣的是,在缺乏Doa4p或Vps21p的细胞中，游离泛素的数量减少，泛素的过表达恢复了vps9Δ细胞中的缺陷货物内化，这表明vps9Δ细胞中的PM完整性缺陷是由缺乏游离泛素引起的。
