Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells

Abstract

Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines. Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells. Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein. Butanol interfered with both the endoplasmic reticulum to Golgi complex transport of the caseins and secretory vesicle formation from the trans-Golgi network. In contrast, the transport of whey acidic protein to the Golgi was less affected. Activation of protein kinase C enhanced the overall secretion of both markers and interestingly, this stimulation of secretion was maintained for whey acidic protein in the presence of butanol. Transphosphatidylation assays demonstrated the existence of a constitutive phospholipase D activity which was stimulated by the activation of protein kinase C. We conclude that phospholipase D plays a role in casein transport from the endoplasmic reticulum to the Golgi and in the secretory vesicle formation from the trans-Golgi network. Moreover, our results suggest a differential requirement for phospholipase D in the secretion of caseins and that of whey acidic protein.

αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

“…In line with this, it should be noted that our permeabilisation experiments were performed at 4°C to avoid proteolysis during incubation. Notably, it has been demonstrated that the phosphorylation of β-casein occurs in a later Golgi compartment than that of α S1 -casein [16-18]. Moreover, since bovine non-phosphorylated β-casein was shown to have a different micellisation process than the phosphorylated form [42], we can hypothesise that the mature and immature forms of rat β-casein have different association properties.…”

“…Phosphorylation allows calcium phosphate binding and further interactions between caseins, a key step in the formation of casein micelles. Notably, the phosphorylation of β-casein seems delayed compared to that of α S1 -casein [16-18]. This suggests that strong interaction of this protein with casein polymers might be postponed until it is trafficked to trans Golgi cisternae.…”

Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice

“…The sequences of a-(aS1 in ruminants) b-, and ccasein (aS2-casein in ruminants) contain one or more clusters of phosphorylated residues known as phosphate centres. Phosphate centres can sequester amorphous calcium phosphate, probably in the Golgi vesicles of mammary secretory cells, to form thermodynamically stable complexes of defined chemical composition which are then secreted through the apical membrane of the mammary epithelial cells [10,11,12,13,14]. Milk, like many other biological fluids, is supersaturated with respect to the crystalline mineral of bones and teeth (apatite) but due to the sequestration reaction, is under-saturated with respect to amorphous calcium phosphate.…”

Phospholipase D—Structure, regulation and function

“…Thus, secretion in mast cells, mammary epithelial cells, platelets, neutrophils and HL60 cells is inhibited by the addition of primary alcohols (Fig. 23, Stutchfield & Cockcroft 1993;Gruchalla et al 1990;Lin et al 1991;Benistant & Rubin 1990;Yuli et al 1982;Fensome et al 1996;Brown et al 1998;Siddhanta et al 2000;Way et al 2000;Boisgard & Chanat 2000). However, this could be due to changes other than inhibition of PA formation by PLD.…”