Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.
Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.
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DOI：
10.1091/mbc.e13-03-0160
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发表时间：
2013-06
影响因子：
3.3
通讯作者：
Watanabe R
中科院分区：
文献类型：
--
作者：
Castillon GA;Michon L;Watanabe R
C末端标记的PGAP3的过表达导致MDCK细胞中主要产生溶解的GPI-APs。在这些细胞中，产生的溶解GPI-AP不会被结合到耐洗涤剂的膜中，但仍然是顶端输送的。完全重塑的和溶解的GPI-APs的顶端运输对胆固醇耗竭和N-糖基化的消融都很敏感。大多数糖基化磷脂酰肌醇锚定蛋白(GPI-APs)位于上皮细胞的顶端。GPI-AP的顶端递送被认为是它们与脂筏相关的结果。我们发现，在Madin-Darby犬肾细胞中，C末端标记的PGAP3的过表达导致了GPI-APs的主要产生，这是GPI脂质重塑过程中的中间前体。在这些细胞中，产生的溶解GPI-AP不被结合到耐洗涤剂膜(DRM)中，但仍然被顶部递送，这表明GPI-AP与DRM的结合对于根尖靶向来说是不必要的。相反，完全重塑和裂解形式的GPI-APs的顶端运输都依赖于N-糖基化，证实了N-葡聚糖在顶端蛋白运输中的一般作用。我们还发现，胆固醇的耗尽不仅导致完全重塑的GPI-AP，而且还导致溶质GPI-AP，以及通常靶向于根尖膜的内源可溶性和跨膜蛋白的根尖到基侧的重定向。这些发现证实了胆固醇在顶端蛋白靶向中的重要作用，并进一步证明了胆固醇依赖的顶端分选机制与GPI-AP的DRM关联无关。
Overexpression of C-terminally tagged PGAP3 causes predominant production of lysoGPI-APs in MDCK cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes but are still delivered apically. The apical transport of both fully remodeled and lysoGPI-APs is sensitive to cholesterol depletion and ablation of N-glycosylation. Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.