Abstract
In order to characterize the function of the COOH-terminal (c) region of eukaryotic signal peptides, a 14-amino acid long segment was deleted from a secreted rat liver and intestinal protein, preapolipoprotein-A-IV. This deletion spanned the c-region plus all potential signal peptidase cleavage sites. The functional consequences of this mutation were assessed using an in vitro transcription/translation/microsomal membrane processing system. Removal of these residues had no effect on interaction of the nascent preprotein with signal recognition particle as measured by a translational arrest assay. Although no signal peptidase cleavage of the mutant was detected, the efficiency of its co-translational translocation was similar to the wild type protein. A postinitiation translocation assay was utilized to compare the translocation capabilities of nascent wild type and mutant proteins as their chain lengths were progressively increased. No difference was detected between the two species suggesting that their initial conformations are functionally equivalent as measured by the translocation machinery. We conclude from these studies of preapolipoprotein-A-IV that (i) the efficiency of translocation is not dependent on signal peptidase cleavage and (ii) structural features present in the c-region of the signal peptide are not necessary for interaction with signal recognition particle or for subsequent targeting to, and translocation across, microsomal membranes.
Highlights
From the Departments of $Biological Chemistry and 1)Medicine, Washington University School of Medicine, St
Inorder to characterize the function of the COOH- (SRP) recognizes the signal peptide as it emerges from the terminal (c) region of eukaryotic signalpeptides, a 14- ribosome [2], and directs the nascent protein-ribosome comamino acid long segmentwas deleted from a secreted plex to theER
When salt-washed canine pancreatic microsomal membranes according to translocationand processing of the wild typeand mutant preproteins were analyzed in uitro, we found that thedeletion did not disrupt SRP interaction or the efficiency of translocation even though no cleavage of themutant by signal
Summary
Materials and Radiochemicals”SP6 polymerase, RNasin, and nu- cell free system, further translation initiation was blocked byadding clease-treated reticulocyte lysate were from Promega Biotec. Positions 5-15 of the rat preapoA-IV signal peptide sites in the recombinant plasmid (Fig. l), digestion with these enzymes was used to confirm that the deletion had been successfully accomplished (data not shown). Digestion with BalI was performed to check that this site hadbeen “regenerated” conserving the two codons which flank the deletion (i.e. GCC and AAT in Fig. contain a block of hydrophobic residues which are predicted by the rules of Chou and Fasman [42] to have a high probability for assuming a P-sheet structure (Fig. 2, panels A and B ). Contain a block of hydrophobic residues which are predicted by the rules of Chou and Fasman [42] to have a high probability for assuming a P-sheet structure (Fig. 2, panels A and B ) This area represents a typical eukaryotic signal peptide.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
