Breuza L, Monlauzeur L, Arsanto J P, Le Bivic A
Laboratoire de Génetique et Physiologie du Développement, Faculté des Sciences de Luminy, Marseille.
J Soc Biol. 1999;193(2):131-4.
In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.
在上皮细胞中，质膜被划分为生化和功能不同的结构域。例如，在肠细胞中，顶端结构域面向肠腔，参与营养物质的摄取，而基底外侧结构域则介导细胞间粘附和信号传导。我们感兴趣的是破解这些特殊结构域形成和维持的潜在机制。作为上皮模型，我们使用了肠细胞系Caco-2，并研究了人类神经营养因子受体（p75 NTR）在这些细胞中的转运和分选。新合成的p75 NTR首先被转运到基底外侧膜，然后在转胞吞作用后积累在顶端膜上。这种最终的顶端定位受膜锚定物和位于靠近膜的胞外结构域部分的O-糖基化位点簇的控制。在可能参与顶端成分分选的机制中，我们寻找了高尔基体中脂-蛋白微结构域形成的作用。这些膜微结构域高度富集糖基磷脂酰肌醇（GPI）锚定蛋白、糖鞘脂和顶端蛋白，如蔗糖酶异麦芽糖酶（SI）。这种组成也存在于称为小窝的内吞结构中，小窝由小窝蛋白1构成。我们在不表达小窝蛋白1的Caco-2细胞中表达了小窝蛋白1，还表达了功能未知的相关蛋白小窝蛋白2。小窝蛋白1的表达导致小窝的形成，表明该蛋白是小窝形成所必需的，而小窝蛋白2局限于高尔基体，对小窝形成没有影响。然而，小窝蛋白2增加了微结构域中SI的掺入量，表明其在招募进入顶端途径中发挥作用。运输小泡融合位点的选择是胞吐作用中控制的最后一步。为了鉴定参与该步骤的蛋白质，我们克隆并表征了t-SNARE家族的两个成员，即Syntaxin 3和SNAP23。Syntaxin 3存在于顶端膜上，并与也位于基底外侧膜上的SNAP23形成复合物，在基底外侧膜上它与Syntaxin 4形成复合物。在Caco-2细胞中过表达Syntaxin 3导致SI向顶端膜的胞吐作用减少，证实Syntaxin 3参与将顶端运输小泡的融合靶向细胞的顶端极。
