title: Translocation of globin fusion proteins across the endoplasmic reticulum membrane in Xenopus laevis oocytes.
abstract: We have studied the translocation of a normally cytoplasmic protein domain across the membrane of the endoplasmic reticulum in cell-free systems and in Xenopus laevis oocytes. Coding regions for the normally cytoplasmic protein globin were engineered in frame either 3' or 5' to the coding region for the signal sequence of either Escherichia coli b-lactamase or bovine preprolactin, respectively, in SP6 expression plasmids. RNA transcribed from these plasmids was microinjected into oocytes as well as translated in cell-free systems. We demonstrate that both in vivo and in vitro, a previously amino-terminal signal sequence can direct translocation of domains engineered to either side. Moreover, the domain preceding the signal sequence can be as large as that which follows it. While, in general, cell-free systems were found to faithfully reflect translocation events in vivo, our results suggest that a mechanism for clearance of signal peptides after cleavage is present in intact cells that is not reconstituted in cell-free systems.
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014982:Xenopus laevis
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005914:Globins