1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

Secretory proteins made in Xenopus luevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two-dimensional gel electrophoresis and by concanavalin-A -Sepharose binding from the cytosolic form, is still secreted. Guinea-pig milk proteins and human interferon are also exported from tunicamycin-treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion, and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.Oocytes of Xenopus luevis will export guinea-pig caseins and human interferon encoded by microinjected mRNAs; heterologous non-secretory proteins are not secreted [l]. Recently we have shown that secretory proteins from such diverse sources as rats, insects and plants are also selectively exported from oocytes (Lane et al., unpublished observations). These results demonstrate the possible usefulness of the oocyte as a general system for studies of protein secretion. In this paper we further examine the fidelity of protein secretion in this system by correlating the subcellular location and secondary modification of secretory proteins with their subsequent fate.Nascent polypeptide chains of secretory proteins contain a 'signal' sequence of some 15-30 amino acids [2-51, which interacts with a putative membrane receptor. This interaction results in the vectorial discharge of nascent polypeptides into the lumen of the endoplasmic reticulum. Secondary modification (e.g. glycosylation [6], phosphorylation [6]) of protein can occur within the endoplasmic reticulum and the use of tunicamycin, an inhibitor of glycosylation [7,8] indicates that this modification may protect proteins from degradation [9], affect the rate of secretion [lo, 111 or alter the intracellular destination of proteins [12]. The subsequent events in secretion are less well understood but are popularly thought Ahhreviution. PhMeSOZF, phenylmethylsulfonyl fluoride.to involve encapsulation of secretory polypeptides within the Golgi apparatus followed by movement of the resultant secretory vesicles to the cell surface, where exocytosis occurs [6,13,14].It is generally believed that the co-translational transfer of secretory proteins across the membranes of the endoplasmic reticulum is a mandatory step in the secretion of these proteins [3,4] ; the corollary of this theory is that proteins localized in the cytosol cannot be secreted. Much of...

A serinc-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated M , 74000 as judged by sodium dodecyl sulphate/polyacrylamidc gel electrophoresis, compared with a monomer M , of 50 000 as determined by sucrose gradient centrifugation in the presence of500 mM NaCl and 0.1 %, Triton X-1 00. Kinetic studics show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and 11, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissuc. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Colgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, i n this instance the Golgi apparatus.We have previously dcscribed the presence in the lactating guinea-pig mainmary gland of a serine-specific casein kinasc, an integral membrane protein located in a Golgi-enriched membrane fraction [l]. Similar enzyme activities have been described in Golgi-enriched fractions isolated from mammary glands of othcr species [2-41. Other casein kinase activities isolated from diverse tissues and cells have also been described in the literature (see [S]). These are CAMP-independcnt and arc classified on the basis of their elution from DEAE-cellulose a s casein kinases 1 and 11; they differ from each other with respect to iiucleotide triphosphate substrate. Casein kinase 11, utilises both ATP and GTP [6] whikt casein kinase I has a preference for ATP. In additioi. cascin kinase I1 but not casein kiiiase I is inhibited by h e p r i n [7].Studies on the phosphorylation of guinea-pig caseins using lactating mammary gland explants in culture [8] or Xrnopus oocytes microinjected with milk protein mRNA [9] demonstrate that each contain partially processed unphosphorylatcd caseins sequestered within the secretory pathway, but that only those secreted by the guinea-pig mammary explants, not by the oocyte, are phosphorylated. These observations suggest that casein kinase is not an enzyme common to the secretory pathway of all cells, and may therefore be expressed in a tissue-specific manner.To determine whet...