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Rôle des microdomaines membranaires dans le ciblage apical de la nucléotide pyrophosphatase NPP3 dans les cellules MDCK

Abstract : Summary The plasmic membrane of the polarized epithelial cells comprises two distinct domains, the apical domain and the basolateral domain. Each domain has a determined composition in lipids and proteins, enabling them to ensure of the specific functions. The molecular mechanisms responsible for the sorting and the targeting of transmembrane proteins towards the apical pole are still badly known. The apical membrane is enriched in glycosphingolipides and cholesterol which form microdomains called “rafts”. In experiments, the rafts can be isolated in the form of DRM (detergent-resistant membranes) defined by their resistance to a nonionic detergent, the Triton X-100. It was proposed that the rafts recruit apical proteins on the level of the trans-golgien network and are used as platform for their targeting with the apical pole. Indeed the proteins anchored by the glycosylphosphatidyl-inositol are resistant to the Triton and are in general localised with the apical membrane. On the other hand, the majority of apical transmembrane proteins are soluble in the Triton, although they are resistant to the action of mild detergents like Lubrol WX. The objective of work of thesis was to study the role of the rafts in the apical transmembrane protein targeting and to include/understand the differential effect of the Triton and Lubrol on their solubilization. The nucleotides pyrophosphatases NPP1 (basolateral) and NPP3 (apical) expressed in a stable way in cells MDCK were used as models. NPP3 is insoluble in Lubrol and partially insoluble in the Triton, while NPP1 is primarily solubilized. The study of the localization and the sensitivity to the detergents of mutants and chimeraes a combining of the cytoplasmic, transmembrane and extracellular domains of NPP3 and NPP1, showed that there was not strict correlation between apical targeting and resistance to the detergents. The resistance of NPP3 to solubilization by Lubrol is acquired precociously during its biosynthesis, independently of its final destination. This resistance depends on amino acids charged positively located in the cytoplasmic tail, close to the membrane. In order to include/understand the selectivity of the Triton and Lubrol in the extraction of proteins and the membrane lipids, the lipidic composition of the DRM obtained after extraction by the Triton and Lubrol were compared. The DRM extracted by the Triton and Lubrol are cholesterol enriched what corresponds to the definition of the rafts. However, the DRM Triton are impoverished in lipids of the internal layer while the DRM Lubrol are enriched in phosphatidylethanolamine. The DRM Lubrol are also enriched in protein associated with the internal layer with the membrane. In conclusion, this work shows that the resistance of apical protein NPP3 to the extraction by Lubrol, and partly by the Triton, is an intrinsic property which probably corresponds to an adaptation of protein to the lipidic composition of the apical domain, but that this property does not determine its polarized targeting. Moreover, this work shows that the detergents are very interesting tools to study the interactions between the membrane proteins and lipids, but that there probably does not exist detergent able to insulate in a strict way of the membrane microdomains such as are defined the rafts. Our results suggest that the layer interns rafts is enriched in phosphatidylethanolamine and cholesterol, that it is partly solubilized by the Triton, which would destabilize transmembrane proteins and would involve their extraction. Keywords : detergent, raft,, apical targeting, cholesterol, membrane microdomain, inner membrane leaflet, phosphatidylethanolamine.
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Contributor : Jean-Louis Delaunay <>
Submitted on : Thursday, November 5, 2009 - 5:47:07 PM
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  • HAL Id : tel-00429983, version 1


Jean-Louis Delaunay. Rôle des microdomaines membranaires dans le ciblage apical de la nucléotide pyrophosphatase NPP3 dans les cellules MDCK. Biologie cellulaire. Université Pierre et Marie Curie - Paris VI, 2007. Français. ⟨tel-00429983⟩



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