Single-Amino-Acid Substitutions within the Signal Sequence of Yeast Prepro-α-Factor Affect Membrane Translocation

Abstract

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast α-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the α-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact α-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-α-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of α-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.