The membrane-associated form of as1- casein interacts with cholesterol-rich detergent-resistant microdomains
2014
Le Parc, Annabelle | Honvo Houeto, Edith | Pigat, Natascha | Chat, Sophie | Léonil, Joelle | Chanat, Eric | Unité de recherche génomique et physiologie de la lactation (GPL) ; Institut National de la Recherche Agronomique (INRA) | Foods for Health Institute, Department of Food Science and Technology ; University of California [Davis] (UC Davis) ; University of California (UC)-University of California (UC) | Génétique Animale et Biologie Intégrative (GABI) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | Centre de recherche Croissance et signalisation (UMR_S 845) ; Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | Université Paris Descartes - Faculté de Médecine (UPD5 Médecine) ; Université Paris Descartes - Paris 5 (UPD5) | Science et Technologie du Lait et de l'Oeuf (STLO) ; Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST
Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that as1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of as1-casein in rat mammary epithelial cells. Using metabolic labelling we show that as1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of as1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of as1-casein. These experiments reveal that the insolubility of as1-casein reflects its partial association with a cholesterolrich detergent-resistant microdomain. We propose that the membrane-associated form of as1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.
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