Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.
The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.
Allison
D S
DS
Department of Biochemistry, University of Washington, Seattle 98195.
Young
E T
ET
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase