A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains.
To determine whether a functional amino terminal signal sequence can be active at an internal position, a hybrid gene was constructed in which the entire coding region of bovine preprolactin cDNA was inserted into chimpanzee alpha-globin cDNA 109 codons downstream from the initiation codon of globin. When RNA synthesized in vitro from this plasmid (pSPGP1) was translated in the rabbit reticulocyte cell-free system, a 32-kD protein was produced that was both prolactin and globin immunoreactive. When microsomal membranes were present during translation (but not when added posttranslationally), a 26-kD and a 14-kD product were also observed. By immunoreactivity and electrophoretic mobility, the 26-kD protein was identical to mature prolactin, and the 14-kD protein appeared to be the globin domain with the prolactin signal sequence attached at its carboxy terminus. From (a) posttranslational proteolysis in the presence and absence of detergent, (b) sedimentation of vesicles in the presence and absence of sodium carbonate pH 11.5, and (c) N-linked glycosylation of the globin-immunoreactive fragment after insertion of an Asn-X-Ser N-linked glycosylation site into the globin coding region of pSPGP1, it appears that all of the 26-kD and some of the 14-kD products, but none of the 32-kD precursor, have been translocated to the lumen of the membrane vesicles. Thus, when engineered to an internal position, the prolactin signal sequence is able to translocate both flanking protein domains. These data have implications for the understanding of translocation of proteins across the membrane of the endoplasmic reticulum.
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005818:Genetic Engineering; D005914:Globins / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D010957:Plasmids; D011388:Prolactin / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*
D006023:Glycoproteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011506:Proteins; C017671:preprolactin; D011388:Prolactin; D005914:Globins