Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.
We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.
D000818:Animals; D001389:Azirines / Q000494:pharmacology*; D001565:Benzoates; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism; D010944:Plants / Q000378:metabolism; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
D001389:Azirines; D001565:Benzoates; D003432:Cross-Linking Reagents; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012346:RNA, Transfer, Amino Acyl; C017671:preprolactin; C051320:4-(3-trifluoromethyldiazirino)benzoic acid; D011388:Prolactin; D003513:Cycloheximide