Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.
The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.
Tokunaga
M
M
Laboratories of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kawamura
A
A
Omori
A
A
Hishinuma
F
F
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D055646:Killer Factors, Yeast; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009183:Mycotoxins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011233:Precipitin Tests; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; C527157:KHR1 protein, S cerevisiae; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000516:alpha-Amylases