National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki305-8642, 2 Japan Women's University, Faculty of Home Economics,2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, 3 Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874 and 5 Water Research Institute, 2-1-6, Sengen, Tsukuba, Ibaraki 305-0047, Japan
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Abstract |
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Keywords: ß-sheet structure/killer toxin/toxic gene
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Introduction |
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A variant SMKT with higher stability and at the same time showing higher killer activity is now being sought in view of the need for further elucidation of the structurefunction relationship of SMKT and the potential use of the toxin in a variety of fields. To accomplish this goal, a suitable system for expression of SMK1 is indispensable. However, despite several attempts to establish an SMK1 expression system, expression of active SMKT has not yet been achieved.
In the case of the K1 toxin of S.cerevisiae, the immunity is determined by the toxin precursor molecule, and expression of the K1 killer gene confers immunity (Boone et al., 1986; Zhu and Bussey, 1991
). On the other hand, expression of the
subunit of Kluyveromyces lactis toxin results in death of the host cells. However, this lethal effect was found to be prevented by expression of the killer immunity gene, which is specified as ORF3 of the linear plasmid pGKL1 of K.lactis (Tokunaga et al., 1989
). By analysis of resistant mutants, it was suggested that intracellular expression of the
subunit mimics treatment with exogenous toxin (Butler et al., 1991
).
In this study, we show that expression of SMK1 under the control of a galactose-inducible promoter is lethal in S.cerevisiae. We also determined the SMK1 regions required for the lethality in S.cerevisiae. The roles of each polypeptide module defined by the crystal structure are discussed in terms of their lethality in S.cerevisiae.
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Materials and methods |
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Saccharomyces cerevisiae W303-1A (MATa, ade2, his3, leu2, trp1, ura3, can1) and CS701C (spf1::LEU2 in W303-1A background) were used throughout this study. YPD contained 1% yeast extract, 2% peptone and 2% glucose and YPD/MB was YPD containing 0.003% methylene blue and 2% agar. Selective media contained 0.67% yeast nitrogen base (Difco, USA), 2% glucose and appropriate supplements as needed. YPGal contained 1% yeast extract, 2% peptone, 2% galactose and 25 mM citrate-phosphate buffer (pH 3.5). YPGal/MB plates contained 1% yeast extract, 2% peptone, 2% galactose, 0.003% methylene blue and 2% agar.
Methods
SMK1 and SMK1 derivatives were inserted into pYES2 (Invitrogen, USA) containing the Gal1 promoter (pGal) and the terminator for expression, and URA3 and 2µ sequences for selection and replication in yeast. Yeast transformation was performed by electroporation (Becker and Guarente, 1991). Standard molecular manipulations were as described by Sambrook et al. (1989). Site-directed mutagenesis was performed using the QuickChangeTM mutagenesis kit (Stratagene, USA) according to the manufacturer's instructions. The positive clones were verified by DNA sequencing using a BigDye terminator sequence kit (Perkin Elmer) and specific primers. In analysis of the expressed SMK1 product, transformants were cultured in 10 ml selective medium overnight. The cells were collected by centrifugation, resuspended in 2 ml YPGal and cultured overnight. Preparation of cell lysates, precipitation of secreted proteins by TCA, electrophoresis and immunoblotting were performed as described previously (Suzuki, 1999
).
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Results and discussion |
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Using the methylene blue plate assay, the lethal effect of modified SMK1 was examined (Figure 1A). Substitution of the
-factor signal sequence for the original signal sequence of SMK1 (pCS204), or deletion of the
peptide region (p
), did not affect the lethality. However, deletion of the signal peptide region (p
Sig) resulted in the loss of lethality, suggesting that entering the secretory pathway is important for the toxicity. In the case of the K.lactis toxin, expression of the
subunit without a signal sequence is also lethal for the host cells (Tokunaga et al., 1989
). Therefore, the mechanism responsible for the toxicity of SMKT may be different from that in the case of the K.lactis toxin.
Deletion of the 3' region corresponding to 21 amino acid residues at the C-terminus (del39) also resulted in the loss of lethality. In order to determine the amino acid residues in the C-terminal region that are involved in the lethality, a stop codon or an amino acid substitution was introduced into the 3' region of SMK1 by site-directed mutagenesis. The stop codons inserted between Ala208 and the C-terminus (pMK207pMK221) constituting a C-terminal loop had no effect on the lethality. On the other hand, the stop codon insertion at Leu207 (pMK206) resulted in the loss of activity (Figure 1B). To investigate the role of the Leu207 residue, Leu207 was replaced by Ala (pMK207A), Ser (pMK207S) and Glu (pMK207E). Cells expressing the plasmid-encoding genes with the Ala or Ser substitution showed a lighter blue color than those harboring pCS224, whereas the Glu substitution resulted in complete loss of lethality (Figure 1B
). As shown in Figure 2
, Leu207 is located at the C-terminus of the central strand of the ß-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the ß-sheet and the
-helices. The results obtained from the substitutions of Ala, Ser or Glu for Leu207 indicated that the nonpolar side chain of Ala or Ser fits the hydrophobic pocket, but the carboxyl group of Glu destabilizes the structure critical for the lethal effect of the SMK1 product. Thus, the side chain of Leu207 may play a key role in stabilizing the overall structure of the SMK1 product.
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In the case of the K1 toxin and the -factor of S.cerevisiae, association of the precursors with the membrane has been observed and this has been thought to be evidence of post-translational signal cleavage. Most of the endogenous SMK1 precursors were also observed in the microsome fractions but not in the soluble fractions, suggesting that stable association with the membrane occurred. As shown in Figure 3A
, a 15 kDa product of p
corresponding to the
+ß polypeptide was observed in the microsome fraction. A Kex2p site (LysArg) exists between the glycosylated signal sequence and the
+ß polypeptide. Expression of the
+ß polypeptide is evidence that the precursor has reached the Golgi.
The secreted form of proSMKT (ß) in P.farinosa was purified and shown to have no killer activity (Suzuki, 1999
). Considering the C-terminus of the
subunit and the N-terminus of the ß subunit in the crystal structure of SMKT (Figure 2
), the
domain is likely to cover the upper part of SMKT. Because endogenous expression of SMK1 products with the
domain is lethal and proper folding may be required for the lethality, it seems unlikely that the
domain prevents the target-binding site from interacting with the target in sensitive cells. Therefore, the
domain may mask the receptor-binding domain that is only required for the exogenous toxicity. The killer strain of P.farinosa is immune to SMKT. The immunity determinant of SMKT may prevent the protoxin from interacting with its own target. Although the lethality of SMK1 in S.cerevisiae described here is a heterologous system, it serves as a basis for elucidation of the immunity mechanism of SMKT.
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Acknowledgments |
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Notes |
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1 To whom correspondence should be addressed; email: csuzuki{at}nfri.affrc.go.jp
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References |
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Received July 26, 1999; revised October 27, 1999; accepted November 12, 1999.