Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis.
Signal recognition particle (SRP) induces elongation arrest of nascent presecretory proteins as the signal peptide protrudes from the large ribosomal subunit. To examine the relationship between the size of the precursor and extent of SRP mediated inhibition of polypeptide chain elongation, we performed in vitro translation experiments in the presence of SRP using a series of truncated preproinsulin mRNA molecules. These precursors possessed the same NH2 terminus as native preproinsulin followed by progressively shorter COOH termini. SRP inhibited translation of precursors as short as 64 amino acids in length, however, the extent of inhibition diminished for shorter precursors. This correlated with a reduction in the time required for ribosomes to transit through the mRNA encoding the shortened precursors. By exploiting a chimeric protein comprising the first 71 residues of preproinsulin fused to the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase, we demonstrate that the largest size a nascent chain can reach and still be susceptible to SRP-mediated elongation arrest is approximately 17 kDa. Our data support the model that SRP binding to the signal peptide is a reversible process even in the absence of microsomal membranes, and that SRP can arrest polypeptide chain elongation at multiple stages during translation.
Okun
M M
MM
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Shields
D
D
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005399:Fishes; D007328:Insulin; D010957:Plasmids; D011384:Proinsulin / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000494:pharmacology*; D018271:Signal Recognition Particle
D007328:Insulin; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; C013737:preproinsulin; D011384:Proinsulin