Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae.
Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.
Suzuki
C
C
National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. csuzuki@nfri.affrc.go.jp
Kawano
M
M
Kashiwagi
T
T
Arata
Y
Y
Kawasumi
T
T
Kashiwagi
Y
Y
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D019943:Amino Acid Substitution; D002470:Cell Survival / Q000187:drug effects; D018388:Codon, Terminator; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000633:toxicity; D005690:Galactose / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000633:toxicity; D015151:Immunoblotting; D007202:Indicators and Reagents; D055646:Killer Factors, Yeast; D008751:Methylene Blue; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D010843:Pichia / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D018388:Codon, Terminator; D003603:Cytotoxins; D005656:Fungal Proteins; D007202:Indicators and Reagents; D055646:Killer Factors, Yeast; D009183:Mycotoxins; C406793:SPF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; D008751:Methylene Blue; D005690:Galactose