The role of the hydrophobic domain in orienting natural signal sequences within the ER membrane

The orientation of signal sequences during insertion into the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar domain. Using recombinant and mutant proteins, also length and hydrophobicity of the apolar segment were shown to affect the orientation: translocation of the N-terminus was found to be favored by long hydrophobic sequences, and translocation of the C-terminus, by short ones. Here, we tested the physiological significance of this phenomenon by mutagenesis of the hydrophobic portion of two natural signals with unusual flanking charges. Extending the hydrophobic domain of the short, cleaved Ncyt/Cexo signal of pre-provasopressin-neurophysin II and shortening that of the Nexo/Ccyt signal anchor of microsomal epoxide hydrolase resulted in a significant fraction of polypeptides inserting in the opposite orientation to that of the wild-type proteins. The topogenic contribution of the hydrophobic domain is thus important for the correct and uniform orientation of natural proteins and can explain the behavior of some of the signals with unusual flanking charges.