Detergent-insoluble GPI-anchored proteins are apically sorted in Fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting
Detergent-insoluble GPI-anchored proteins are apically sorted in Fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting
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DOI：
10.1091/mbc.11.2.531
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发表时间：
2000-02-01
影响因子：
3.3
通讯作者：
Zurzolo, C
中科院分区：
文献类型：
--
作者：
Lipardi, C;Nitsch, L;Zurzolo, C
与Madin-Darby犬肾细胞相比，Fischer大鼠甲状腺细胞将大部分内源性糖基磷脂酰肌醇（GPI）锚定蛋白递送到基底外侧表面。然而，我们在这里报道了GPI蛋白胎盘碱性磷酸酶（PLAP）和神经营养因子受体-胎盘碱性磷酸酶（NTR-PLAP）在转染的Fischer大鼠甲状腺细胞中顶端定位。与“筏假说”一致，假设GPI蛋白与鞘糖脂和富含胆固醇的筏结合，我们发现这两种蛋白质都不溶于Triton X-100，漂浮在蔗糖密度梯度的较轻部分。然而，去除胆固醇对脂筏的破坏并没有引起皮瓣和NTR-LAP表面的错误分类，并且改变了。伏马菌素B1处理后这些蛋白的表面分选与Triton X-100不溶性组分水平的降低无关。此外，与这两种蛋白的gpi锚定形式相反，分泌和跨膜形式（在没有基底侧细胞质信号的情况下）被分类到顶端表面，而与脂质微结构域无关。综上所述，这些数据表明，GPI锚需要调解筏关联，但不足以确定顶端排序。他们还认为，存在于蛋白质外域的信号起着主要作用，脂筏可能促进对反式高尔基网络中这些信号的识别，即使它们不是典型分类所必需的。
In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous,glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into,glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of FLAP and NTR-LAP, and the altered. surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for epical sorting.