Abstract
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 αs1-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 αs1-casein in rat mammary epithelial cells. Using metabolic labelling we show that αs1-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 αs1-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 αs1-casein. These experiments reveal that the insolubility of αs1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of αs1-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.
Highlights
During lactation, the mammary epithelial cells (MECs) synthesise and secrete substantial quantities of milk-specific proteins and other components such as lipids and lactose in a polarised fashion, from their apical surface into the alveolar lumen that they surround
The main milk proteins are the caseins, a family of acidic phosphoproteins. During their transport through the secretory pathway, caseins interact with calcium and calcium phosphate, and progressively self-aggregate to organize into a supramolecular structure, the casein micelle, which is released by exocytosis into the milk
We further investigate the molecular basis of as1-casein association with membranes of the secretory pathway, with emphasis on its potential incorporation into detergent-resistant membranes (DRMs) microdomains [18], an interaction which may be a prerequisite for the formation, transport and sorting of casein micelle to the apical plasma membrane domain of the MEC
Summary
The mammary epithelial cells (MECs) synthesise and secrete substantial quantities of milk-specific proteins and other components such as lipids and lactose in a polarised fashion, from their apical surface into the alveolar lumen that they surround. The hypothesis that caseins would be clustered into small spherical subunits that would be further linked together by calcium phosphate was widely accepted This theory led to the submicelle model of the internal structure of the casein micelle. Models that refute the concept of discrete subunits within the casein micelle have emerged One of these is the tangled web model, first proposed by Holt [4], and extended by Horne [5]. The detailed intrinsic organisation and the mechanisms involved in the formation of this structure have not been fully established This is not trivial since it is well known that the mesostructure of the micelle determines the techno-functional characteristics of the milk protein fraction and impacts milk processing
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