The Influence of Topology and Glycosylation on the Fate of Heterologous Secretory Proteins Made in Xenopus Oocytes

Abstract

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proteins from such diverse sources as rats, insects and plants are also selectively exported from oocytes (Lane et al., unpublished observations).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...

Characterisation of a Membrane‐Bound Serine‐Specific Casein Kinase Isolated from Lactating Guinea‐Pig Mammary Gland

“…Both approaches demonstrate that the synthesis of membrane and secretory proteins is tightly coupled to sequestration and core glycosylation at the level of the endoplasmic reticulum, and that these mechanisms are common to many cell types [ 5 ] . Recent studies on the mechanisms of secretion using Xenopus oocytes also demonstrate that intracellular events subsequent to sequestration must be common to different cell types, and that the posttranslational addition of prosthetic groups, such as the glycosylation of known glycoproteins, is not required to ensure secretion [6].…”

In Vitro Synthesis and Processing of Wheat α-Amylase

“…Glycosylation by the oocyte system can be inhibited with tunicamycin, an antibiotic which inhibits asparagine-dependent glycosylation. Colman et al (8) found that tunicamycin-treated oocytes produced a smaller form of ovalbumin than is found in vivo. A similar result has been obtained with the G I storage protein of bean.…”

“…Usually, glycosylation accompanies cleavage of the signal peptide, but as has been shown by Bielinska, Blobel, and Colman, cleavage can occur when glycosylation is inhibited (2,6,8). Prevention of glycosylation in vitro has been accomplished by isolating signal peptidase activity from membranes by treatment with detergent.…”

“…Even if wheat a-amylase were glycosylated at the level seen for the barley protein, we would probably not detect the 290-dalton difference with our gel system. Lane et al (8,23) have shown that oocytes programmed with heterologous RNA from secretory proteins are able to translate, process, and secrete these proteins into the incubation medium.…”

Signal sequences, secondary modification and the turnover of miscompartmentalized secretory proteins in Xenopus oocytes

“…The half-lives recorded (Table 1) The influence of secondary modification of protein stability can be studied using pairs of proteins differing only in, for example, their degree of N-glycosylation or phosphorylation. Tunicamycin-treated oocytes [8] Fig. 1 C: in the experiment shown in B exported frog proteins are swamped by chicken proteins.)…”