Parallel effects of signal peptide hydrophobic core modifications on cotranslational translocation and posttranslational cleavage by purified signal peptidase

Secretory proteins are targeted to the secretory pathway by an amino terminal extension sequence known as the signal peptide. Signal peptides contain a central core of uncharged amino acids that are usually hydrophobic in nature. The hydrophobic core of the parathyroid hormone (PTH) signal peptide is composed of 12 contiguous hydrophobic amino acids. Although the exact role of the core in signal peptide function is not well defined, particularly in mammalian systems, its length and hydrophobicity have been shown to be important characteristics. To determine the requirements for length and hydrophobicity of a mammalian hydrophobic core, amino acids were substituted and deleted from this region of the PTH signal peptide and the effects on protein translocation and processing were examined in a mammalian cell-free system. Increasing the length of the core by 3 residues had little effect on translocation and processing of the mutant signal peptide by microsomal membranes. Decreasing the length of the core by 6 residues abolished signal activity and partially blocked its interaction with signal recognition particle. Deleting only 2 residues from the core resulted in a position dependent effect on translocation and processing by microsomal membranes that was unrelated to core hydrophobicity but correlated inversely with core amphiphilicity. Deletion at the N-terminus of the core caused a significant decrease in signal activity while deletions at the C-terminus had little effect. Signal activity improved as the position of the pair-wise deletion moved from the N-terminus to the C-terminus of the core. Precursor proteins containing defective signal peptides were unable to translocate across the microsomal membrane. Parallel effects of these modifications on post-translational cleavage by purified signal peptidase were observed. A model is proposed to explain these parallel effects.U of I OnlyETDs are only available to UIUC Users without author permissio