Immunocytochemical Analysis of Pep tide Hormone Processing: Importance of the Positively Charged N-terminal Domain of Signal Peptide in Correct ER Targeting in Yeast Cells

Abstract

We used a morphological approach to determine the topogenic role of the signal peptide in mediating the ER translocation of yeast prepro-α-factor. In prepro-α-factor-somatostatin hybrids, changes in the N-terminal amino acid sequence from wild-type NH₂-Met-Arg-Phe (MRF) to NH₂-Met-Phe-Lys (MFK) caused a subtle difference in protein trafficking in yeast cells (12). Immunofluorescence microscopy on semithin cryosections and immunoelectron microscopy on ultrathin sections showed that the transposition of the charged amino acid at N-terminus caused the precursors to be associated with either nucleus or mitochondria. This suggests that the secretory proteins are mistargeted to the irrelevant organelles as the result of inefficient ER translocation. Structural aspects of nuclear or mitochondrial targeting proteins and common principles in membrane translocation systems account for the mistargeting of overexpressed mutant hybrid precursors that are not rapidly translocated into the ER. Based on our immunocytochemical study on individual cells, we propose here that the positively charged N-terminal domain of signal peptide is important not merely in the efficiency of ER translocation, but also in appropriate targeting of peptide hormone precursors in yeast cells where post-translational ER translocation is known to occur frequently.