Abstract
The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).
Highlights
The carboxyl-terminaslequences of the twopolypep- 1984).This structure has been assigned on the basis of NHptide chainsof the Saccharomyces cerevisiaeK1 killer terminal sequence analysis of the purified a and fi subunits toxin weredetermined by protein sequencing and and of the synthesized in vitro preprotoxin (Bostian et al, amino acid analysis of peptide fragments generated 1984)
Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where thea and j3 subunits consist of amino acid residues 45-147 and 234-316, respectively
The molecular weight of the mature aj3 degree of similarity (Tipper and Bostian,1984).Both require several common activities including the product of the KEX2 gene (Julius et al, 1984).The product of the KEX2 gene has been identified as a dibasic endopeptidase that initially cleaves following pairs of basic amino acids (Julius et al, 1984) and such sitesareapparently operational in both a factor (Julius et al, 1984)and killer toxin maturation (Bostian et al, 1984)
Summary
The carboxyl-terminaslequences of the twopolypep- 1984).This structure has been assigned on the basis of NHptide chainsof the Saccharomyces cerevisiaeK1 killer terminal sequence analysis of the purified a and fi subunits toxin weredetermined by protein sequencing and and of the synthesized in vitro preprotoxin (Bostian et al, amino acid analysis of peptide fragments generated 1984). The COOH-terminal Maturation of yeast a factor and killer toxin displays a high amino acid of the j3 chain is histidine 316, the final residue encodedby the precursor gene. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where thea and j3 subunits consist of amino acid residues 45-147 and 234-316, respectively. The COOH-terminal sequence by the product of the STE13 gene (Julius et al, 1983), and a determination completes the location of the toxin sub- carboxypeptidase B-like activity have been implicated in a units in the precursor, and its configuration may be factor maturation (Fuller et al, 1985). We have determined the sites for the product of the KEX2 gene
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