An N-terminal deletion mutant of preproparathyroid hormone that contains a single basic amino acid, lysine, in the N-terminal domain of the signal peptide is translocated across the endoplasmic reticulum membrane similarly to intact preproparathyroid hormone. To examine the function of charged residues preceeding the hydrophobic core, the lysine was replaced by an uncharged (methionine) or negatively charged (glutamic acid) amino acid. The translocational activity of the mutant signal peptides was assayed in a reticulocyte lysate system containing chicken oviduct microsomal membranes. Altering the net charge of the N-terminal domain did not abolish signal sequence activity, although the efficiency of translocation was decreased for the mutant with a glutamic acid substitution. Posttranslational, ribosome independent, translocation was observed for all the mutants tested, with the same dependence on N-terminal charge but with much lower efficiency than cotranslational translocation. These studies show that the presence of basic amino acids in the N-terminal domain of a eukaryotic signal sequence is not required for its activity.
ASJC Scopus subject areas
- Molecular Biology