Determinants for protein translocation across mammalian endoplasmic reticulum. Membrane insertion of truncated and full-length prelysozyme molecules.
The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full-length prelysozyme using transcription-coupled capping of RNA and subsequent translation in a wheat germ cell-free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal-recognition particles (SRP) to the cell-free system blocked the nascent chain synthesis. The SRP-depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP-induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum.
D000818:Animals; D001692:Biological Transport; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D018271:Signal Recognition Particle
D004792:Enzyme Precursors; D011506:Proteins; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; D004247:DNA; D009113:Muramidase