Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation.
We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-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 alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.
Allison
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DS
Department of Biochemistry, University of Washington, Seattle 98195.
Young
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ET
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D005656:Fungal Proteins / Q000235:genetics*; D007425:Intracellular Membranes / Q000378:metabolism; D000072235:Mating Factor; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005656:Fungal Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor