Intracellular Compartmentation and Secretion of Carp Proinsulin Synthesized in Xenopus Oocytes

Abstract

Carp proinsulin and rabbit globin, synthesized in Xrnopus oocyies in response to simultaneously injected mRNAs, are found in membrane vesicles and in the cytosol respectively. After 24 h of incubation labeled proinsulin, but not globin, is found also in the medium, indicating specific secretion from the oocytes. N o insulin could be found either within or outside the oocytes.The results provide further evidence that the mRNA for a given protein contains the information for its intracellular destination and its eventual secretion.It is generally assumed that the mRNA for a given protein contains the information for its final destination within a cell or for its secretion out of a cell. This information is probably encoded in defined amino acid sequences which are called 'topogenic' sequences [I]. There is some evidence to support this hypothesis. Experiments in vitro have shown that most secretory proteins are synthesized via precursors which contain at their N-termini cleavable hydrophobic signal sequences. These are recognized by a signal receptor in the rough endoplasmic reticulum membrane [2,3] and direct the nascent polypeptide cotranslationally into the lumen of the endoplasmic reticulum membrane [4,5]. It is clear that the signal for the vectorial transport across the membrane is entirely localized in the nascent secretory protein. Accordingly, studies both in viiro and zn vivo have shown that there is no species or tissue specificity of the translocation or processing activity of the endoplasmic reticulum membrane.The further pathway of a secretory protein, which involves its transport to the smooth endoplasmic reticulum, to the Golgi apparatus and finally to the plasma membrane, has not yet been studied successfully in vitro. The Xenopus oocyte system may be a valuable tool for the investigation of these steps in vivo. In Xenopus oocytes foreign eucaryotic mRNAs are translated faithfully after their injection and many cotranslational and posttranslational protein modifications are carried out [6,7]. For example, in Xenopus oocytes the signal peptidase of precursors of some secretory proteins are split off and there is only one report which claimed that human and rat preproinsulins can be detected in oocytes [30]. Furthermore, Lane and his associates have demonstrated for some secretory proteins that the newly made and processed polypeptides are contained within membrane vesicles [I 3,141. Recently, Colman and Morser extended a previous study [I51 and reported the secretion of human interferon and guineapig milk proteins from oocytes into the medium [16].We have previously shown that mRNA isolated from carp islets (Brockmann bodies of Cyprinus carpio) directs the syn- thesis of carp proinsulin in Xenopus oocytes [lo]. In the present paper it is demonstrated that the proinsulin is contained within membrane vesicles. Moreover, after some time it is found in the surrounding medium, On the other hand, newly synthesized rabbit globin is contained within the oocytes in the cyto.;olic compartment and is not s...