The role of caseins in milk protein secretion Deficiency for a-casein severely impairs milk protein secretion in mice. In contrast b-casein deficiency does not result in a similar decline [5] . This suggests that, in contrast to b-casein, a-casein plays a more critical role in the establishment of a functional casein micelle thereby affecting secretion of all casein proteins. Alternatively or additionally due to the higher number of phosphate centres present in a-casein vs. b-casein (3 vs. 1), deficiency for a-casein (but not for b-casein) may decrease the stability of the biofluid resulting in a precipitation of calciumphosphate in the Golgi vesicles. These results argue against a functional redundancy of the calcium-sensitive caseins (a-, b-, cand d-casein). Whereas overall secretion of milk proteins is not adversely affected in heterozygous mice (apart from a 50% reduction in a-casein protein secretion), milk protein secretion from mammary epithelial cells is severely curtailed in homozygous a-casein deficient mice. This affects both, caseins and whey proteins like WAP. In contrast, secretion of albumin which is not secreted by the mammary epithelial cells is not affected by the absence of a-casein. These results suggest a functional role for acasein in casein micelle formation and/or stabilisation (similar to that of k-casein) [6] . We find that the glucose regulated protein 78 (grp78 or BiP) is significantly up-regulated in response to the absence of a-casein. Grp78/BiP is an endoplasmic reticulum (ER)resident protein whose expression is enhanced under conditions of ER stress [29] [31, 33] . Surprisingly the protein is also found in milk of a-casein deficient dams. This suggests that grp78/BiP may be critically involved in the assembly of the casein micelle. One can speculate that grp78/BiP is associated with the maturing casein micelle and is co-secreted with the immature micelle into milk. Two further ER resident proteins, grp94 and PDIA6 are also secreted into milk of a-casein deficient animals indicating that the lack of a-casein has significant impacts on protein processing and transport in the mammary epithelium. Interestingly, secretion of grp78/BiP and grp94 is also observed in mice over-expressing human protein C [37, 38] . This may suggest that perturbation of the balance of milk proteins invokes a common ER stress response resulting in the co-secretion of ER resident proteins into milk. Despite the clear evidence for changes to the expression and localisation of ER resident proteins there is no clear evidence for significant increases in mammary apoptosis in the absence of acasein. Firstly, lactation is not terminated prematurely in the deficient animals suggesting that at least a significant portion of the mammary epithelial cells is viable. Under physiological conditions mammary gland involution after weaning leads to a complete cessation of milk secretion, apoptosis and regression of the epithelial tissue [39, 40] . In homozygous protein C transgenic mice no successful lactation is established, suggesting that in that case ER stress is followed by loss of mammary epithelium function [38] . Secondly, we cannot detect any gross morphological alteration of the secretory mammary epithelium in a-casein deficient mice. Thirdly, we also failed to detect increases in caspase activity in a-casein deficient mammary tissue. Finally, the apoptosis related genes which showed consistent and significant increases in mRNA expression in mammary tissue of a-casein deficient mice include the anti-apoptotic genes survivin (Birc5) and ARC/Nol3, suggesting that the mammary cells are actively avoiding apoptosis. On the other hand a reduction in the expression of the Traf1 gene, encoding an anti-apoptotic gene in the context of the mammary gland [41] suggests a balance of proapoptotic and anti-apoptotic responses to a-casein deficiency. Taken together these findings may suggest that the ER-stress response in mammary epithelial cells of a-casein deficient mice is sufficient to rescue protein secretion, thereby preventing large scale apoptosis and loss of tissue function. Interestingly, we find that steady state levels of mRNAs encoding b-casein, c-casein and k-casein are also severely reduced in the mammary gland of a-casein deficient mice (cf. Fig. 5d ). In contrast no such reduction is observed in heterozygous mice. There could be (at least) two explanations for this. The deletion of cis-acting DNA elements in the a-casein gene may affect the expression of the entire casein gene locus. This genetic explanation would imply that the presence of one functional a-casein allele is sufficient to mediate the activation of both copies of the casein gene locus. Alternatively there may be physiological explanations. ER stress has been associated with an attenuation of protein synthesis [33] . One can speculate that in the mammary gland this leads to a decrease in the transcription of milk protein genes which account for most of the transcripts during lactation to prevent overloading of a compromised endoplasmic reticulum [33] . A decrease in the abundance of milk protein gene specific mRNAs was also observed in the transgenic mice over expressing human protein C [38] . Alternatively, the viability of the secretory mammary epithelial cells may be impaired to some degree by the ER stress response, leading to a lower relative number of casein producing cells in the mammary gland.