Angewandte Molekularbiologie, Universität des Saarlandes, FR 8.3, Gebäude 2, Postfach 151150, D-66041 Saarbrücken, Germany1
Author for correspondence: Manfred J. Schmitt. Tel: +49 681 302 4730. Fax: +49 681 302 4710. e-mail: mjs{at}microbiol.uni-sb.de
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ABSTRACT |
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Keywords: Kex2p endopeptidase, preprotoxin processing, C-terminal HDEL motif
Abbreviations: ER, endoplasmic reticulum; MBA, methylene blue agar; pptox, preprotoxin; SP, signal peptidase
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INTRODUCTION |
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Based on the pptox sequence we previously predicted that the toxin precursor includes an N-terminal signal sequence [necessary for toxin import into the ER (endoplasmic reticulum) lumen] that is cleaved by the action of signal peptidase (SP) most likely after Leu31 or Gly36, producing a 314- or 309-residue precursor, respectively (Schmitt & Tipper, 1995 ). The protoxin resulting from SP cleavage contains an intervening, potentially N-glycosylated
sequence that separates the
and ß subunits. Genetic analysis of pptox gene expression in yeast
kex1 and/or
kex2 null mutants and determination of the N-terminal sequences of the toxins
and ß subunits implied a pattern of processing that strongly resembles prohormone conversion in mammalian cells (Steiner et al., 1992
; Schmitt & Tipper, 1992
; Eisfeld et al., 2000
). In S. cerevisiae, the KEX-encoded prohormone processing machinery is involved not only in the activation of virally encoded killer toxins (such as K1, K2 and K28) but also in the maturation of the yeast pheromone
factor (Dmochowska et al., 1987
; Zhu et al., 1987
; Dignard et al., 1991
; Eisfeld et al., 2000
). Both processing enzymes, endopeptidase Kex2p and carboxypeptidase Kex1p, are membrane-anchored proteins located in a late Golgi compartment and have their N-terminal active sites in the lumen (Fuller et al., 1989
; Redding et al., 1991
). In both mammalian and yeast systems, endopeptidase Kex2p and Kex2p-like convertases such as furin and PC1-PC7 are very similar in their substrate specificity for C-terminal lysine and arginine residues, most often cutting after a sequence of two basic residues (Bryant & Boyd, 1993
; Bevan et al., 1998
). In the case of the K28 toxin precursor, intracellular pptox processing has previously been predicted to be mediated by Kex2p and Kex1p (Schmitt & Tipper, 1995
). The N terminus of
should be produced by cleavage after Glu-Arg49, 18 residues downstream of the predicted SP cleavage site, and the N terminus of ß by Kex2p cleavage after Lys-Arg245. Although the precise C terminus of
has not yet been identified, it has been postulated that it terminates upstream of the first N-glycosylation site (Asn-Ser-Thr163) since the secreted toxin is not glycosylated in vivo. In contrast to the toxins
subunit, the ß C terminus (His-Asp-Glu-Leu344) is generated by the action of yeast carboxypeptidase Kex1p that removes the terminal arginine residue and finally uncovers the toxins ER targeting signal (ß-HDEL), which has recently been shown to be essential for retrograde toxin transport in a sensitive target cell (Eisfeld et al., 2000
).
To extend the in silico predictions for pptox processing, we have now performed site-directed mutagenesis of the pptox gene and analysed the phenotypic effects on K28 toxicity, immunity and toxin secretion in wild-type yeasts as well as in yeast mutants defective in protein precursor processing (kex2 and/or
yps1). We mapped the signal peptidase cleavage site and identified Kex2p sites that are necessary for the in vivo maturation of
and ß. Finally, we show that the two subunits are covalently linked by a single disulfide bond between
-Cys56 and ß-Cys340, and present evidence that the most important in vivo function of Cys340 might be to expose the toxins ß C-terminal ER targeting signal, ensuring accessibility of the HDEL receptor of the target cell.
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METHODS |
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DNA constructs and in vitro mutagenesis.
The K28 killer toxin ORF was isolated by PCR using Taq DNA polymerase and the yeast expression plasmid pPGK-M28-I (Schmitt, 1995 ) as DNA template. Oligonucleotide primers used for PCR amplification were 5'-CCGGAATTCATGGAGAGCGTTTCCTCATTATTT-3' and 5'-CCCAAGCTTTTAGCGTAGCTCATCGTGGCACCT-3', introducing a 5' terminal EcoRI and a 3' terminal HindIII restriction site flanking the toxin-encoding cDNA. The PCR product containing the K28 ORF was cloned between the EcoRI and HindIII restriction sites of yeast multicopy vector pYX242, allowing constitutive expression of a K28 killer phenotype under transcriptional control of the yeast triose phosphate isomerase (TPI) promoter. The Arg
Ala49 mutation was introduced by PCR (splicing by overlapping extension) as previously described by Horton et al. (1989)
using the following primers: 5'-K28, 5'-GAATTCATGGAGAGCGTTTCCTCATTATTTAACATTTTTTC-3'; 3'-K28, 5'-CTCGAGTTAGCGTAGCTCATCGTGGCACCTTG-3'; 5'-R49A, 5'-ATCTGAGAGACAGCAGGGCTTAGAAGAAGCTGACTTCAGTGCTGCTACTTGCGTACT-3' and 3'-R49A, 5'-AGTACGCAAGTAGCAGCACTGAAGTCAGCTTCTTCTAAGCCCTGCTGTCTCTCAGAT-3'. For changing Arg149 into Ala149, primers 5'-K28, 3'-K28 (see above), 5'-R149A (5'-GACTGAAAACGCAGAGAATATACAATCGGCGAGTCTTATACCGGGTCTGCTTAGTAGGGATATAA-3') and 3'-R149A (5'-TTATAATCCATACTAAGCAGACCCGGTATAAGACTAGCCGATTGTATATTCTCTGCGTTTTCAGTC-3') were used. Both constructs were cloned between the EcoRI and XhoI site of pYX242. The double mutant (Ala49/Ala149) was constructed by substitution of the BamHIXhoI fragment of Arg
Ala49 by the corresponding fragment of Arg
Ala149. All other amino acid changes were introduced into the K28 ORF by using the phagemid in vitro mutagenesis kit Muta-Gene Version 2 (Bio-Rad) under conditions recommended by the manufacturer. The sequences of the oligonucleotide primers used to generate mutations within the N-terminal hydrophobic region of pptox were 5'-ATGTCGGCATTCGCCGTGCATATT-3', leading to a change of Gly36 to arginine, and 5'-CGTGCATATTTGGGATTTGAAACA-3' for the exchange of Leu31 to proline. The oligonucleotide primer 5'-ATAAGACTACGCTTTTGTATATTC-3' was used to exchange serine-148 into lysine, thereby conferring the endopeptidase cleavage site predicted to generate the
C terminus into a classical Kex2p site. For an exchange of Cys56 to tryptophan the primer 5'-GCCCATCAGTACCCAAGTAGCAGCA-3' was used. PCR mutagenesis was carried out to change Cys340 to serine with the following oligonucleotides as PCR primers: 5'-CCCAAGCTTTTAGCGTAGCTCATCGTGGGACCTTGCCTCGTCGTC-3' and 5'-CCGGAATTCATGGAGAGCGTTTCCTCATTATTT-3'. Mutated pptox constructs were cloned between the EcoRI and HindIII sites of the yeast expression vector pYX242. Subsequent DNA sequencing of the PCR-amplified K28 pptox fragment identified two bases that always differed from the published K28 sequence (Schmitt & Tipper, 1995
). According to the base positions in the published K28 cDNA sequence, cytosine in position 779 has to be changed into a thymine, and adenine in nucleotide position 981 has to be changed into a thymine. The latter changes codon 256 from the amino acid serine into phenylalanine. Moreover, due to PCR amplification of the wild-type K28 ORF (prior to in vitro mutagenesis), an additional codon change has occurred leading to a change of Arg328 to glycine. However, the strength of the killer phenotype of transformants expressing the killer toxin with the altered sequence was comparable to transformants expressing the wild-type ORF.
Transformation and DNA sequencing.
Wild-type and mutated pptox plasmids were transformed into the indicated yeast strains by the lithium acetate method using salmon sperm DNA as carrier (Schiestl & Gietz, 1989 ). Where indicated, strains BFY113, ME938 and HKY20-11A were co-transformed with YEp6-KEX2 (Zhu et al., 1992
) and maintained on SC-Ura-His medium. YEp6-KEX2 is a 2µ multi-copy plasmid that carries the HIS3 gene and a copy of KEX2 lacking the cytoplasmic C-terminal domain (Zhu et al., 1992
). E. coli strain DH5
was transformed by electroporation using the Gene Pulser II system (Bio-Rad). All DNA constructs used in this work were routinely sequenced by fluorescent cycle sequencing on an automated DNA sequencer (LI-COR 4200, MWG Biotech).
Killer assay and immunity tests.
K28-specific killing and immunity was determined in an agar diffusion assay on methylene-blue agar plates (MBA; pH 4·7) as previously described (Schmitt & Tipper, 1990 ). We used citrate at a final concentration of 2·9% instead of 1·92% as described in the original protocol. Killer strains (about 105 cells in a total volume of 10 µl) were spotted onto MBA plates that had been seeded with an overlay of the sensitive strain S. cerevisiae 192.2d (Schmitt et al., 1996
). After incubating the plates for 4 days at 20 °C, clear zones of growth inhibition surrounding the killer cells (which indicates toxicity) were measured. Killer activity is expressed as the size of the growth inhibition zone. Toxin sensitivity/immunity tests were performed on MBA plates using an overlay of the indicated yeast strain (105 cells per plate) and concentrated culture supernatants of a K28 killer strain as the toxin source. Briefly, concentrated culture supernatants (100 µl) derived from the K28 ski2 mutant S. cerevisiae MS300c (Schmitt & Tipper, 1990
) were pipetted into wells (9 mm diameter) that had been cut into the agar and the plates incubated for 4 days at 20 °C. In this assay, yeast transformants expressing a mutant pptox derivative that is incapable of conferring functional K28 immunity show a sensitive non-killer phenotype, resulting in a cell-free growth inhibition zone around the well.
Yeast cell fractionation.
For subcellular toxin localization, the sensitive yeast strain S. cerevisiae 192.2d was grown at 30 °C in YEPD medium to early exponential phase (1x107 cells ml-1), harvested by centrifugation and used for cell fractionation experiments essentially as previously described (Eisfeld et al., 2000 ). Briefly, yeast spheroplasts were incubated for 1 h at 30 °C in the presence of 104 U purified K28 toxin ml-1. Thereafter, cells were washed in a solution of 0·8 M sorbitol, 20 mM HEPES/KOH, 50 mM potassium acetate and 2 mM EDTA (pH 7·0) and lysed in chilled lysis buffer with the aid of a 5 ml dounce homogenizer (
15 strokes), and the resulting lysate was subjected to differential centrifugation giving four different cell fractions: cell wall fraction (300 g), membrane fraction (plasma-, Golgi- and ER membranes; 13000 g), endosomal vesicle fraction (100000 g pellet) and cytosol (100000 g supernatant).
Western blot analysis.
To estimate the amount of killer toxin secreted by S. cerevisiae after transformation with the various K28 expression vectors, cultures of the appropriate transformants were grown in synthetic minimal medium (pH 4·7) at 30 °C for 2 days until they reached about 5x107 cells ml-1. After centrifugation, 1·2 ml supernatant was ethanol precipitated. The precipitate was dried and redissolved in water for further analysis by SDS-PAGE. If not otherwise stated, SDS-PAGE was performed under non-reducing conditions. Reducing conditions were provided by adding ß-mercaptoethanol to the sample buffer at a final concentration of 6%. Cell lysis was carried out under reducing conditions using 108 cells which were vortexed for 1 min in sample buffer containing an equal volume of acid-washed glass beads. After short-spin centrifugation, the resulting supernatant was analysed by gel electrophoresis. Samples were fractionated either on SDS-polyacrylamide gradient gels (1022·5%) or on Tris/Tricine gels (10%) and electrophoretically blotted onto PVDF membranes in transblot buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0·1% SDS). Western blot analysis of the secreted toxin was carried out using polyclonal antibodies against the toxins and/or ß subunit. Experiments on yeast cell fractionation and subcellular toxin localization were performed as previously described (Eisfeld et al., 2000
).
Protein sequencing.
The secreted pptox processing intermediate in which the two Kex2p sites flanking the N and C termini of had been destroyed (Arg
Ala49, Arg
Ala149) was fractionated by SDS-PAGE, electroblotted onto PVDF membranes and used for N-terminal sequence analysis by Edman degradation as previously described (Schmitt & Tipper, 1995
).
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RESULTS |
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By classical genetics and phenotypic analysis we previously reported that pptox expression in a yeast kex2 null mutant results in immune non-killers completely devoid of secreted toxin activity or protein (Schmitt & Tipper, 1995
). It, therefore, had been concluded that KEX2 function is essential for expression and secretion of the
/ß heterodimeric protein toxin. We now extend these findings by analysing the intracellular K28 processing intermediates in a yeast
kex2 null mutant. Expression of wild-type pptox in this mutant (strain BFY113) resulted in immune non-killers that did not secrete detectable amounts of toxin (Fig. 2a
, lane 2). However, in contrast to the cell-free culture supernatant, intracellular membrane fractions derived from the
kex2 mutant gave a strong anti-ß antibody response and identified a 42 kDa protein species which was absent in the negative control (i.e. in the untransformed mutant; Fig. 2b
, lanes 4 and 5, respectively). Thus, in a
kex2 mutant, the K28 toxin precursor is accumulating within the intracellular membrane fraction most likely within the ER lumen because it is not properly processed. Correspondingly, retransformation of the pptox-expressing
kex2 mutant with a functional copy of KEX2 on the 2µ vector YEp6-KEX2 fully restored pptox processing, resulting in immune killers that secreted significant amounts of a biologically active
/ß toxin (Fig. 2a
, lane 3; see also Table 1
). Interestingly, the 42 kDa protein species seen within the intracellular membrane fraction of a
kex2 mutant perfectly matches the predicted size of the 37·6 kDa toxin precursor after entry into the ER and subsequent core-glycosylation within
: once the toxin precursor has entered the ER lumen, pptox processing should initiate with SP cleavage after Gly36 (see above) and further N-glycosylation should add about 7·5 kDa leading to a protoxin with a calculated size of 41·9 kDa.
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Investigation of potential Kex2p processing sites within and at the N terminus of ß
For the potential Kex2p site within we could show that this sequence (Leu-Tyr-Lys-Arg192) is probably not cleaved in vivo because (as shown above) expression of an Arg
Ala149 toxin derivative resulted in the secretion of a
/
fragment that corresponded exactly to the predicted size of the unprocessed
/
peptide after translocation into the ER, cleavage of the signal peptide and core N-glycosylation of all three potential N-glycosylation sites (Asn-X-Ser/Thr) within
. However, the observed molecular mass of the secreted pptox processing intermediates indicates that the N-linked carbohydrate moiety of all
-containing fragments is probably trimmed back by late Golgi
-mannosidases.
As summarized in Table 1, exactly the same results were obtained after expression of the Arg
Ala149 pptox either in the
kex2 null mutant BFY113 or in the
yps1 mutant HKY20-11A, and no changes were observed after co-expression of Kex2p from YEp6-KEX2 (Table 1
): in each case, besides the predicted and identified
-containing fragments (
/
, 21 kDa;
/
/
, 22·6 kDa), no additional
/
-specific signal was detectable, suggesting that Kex2p does not recognize the internal endopeptidase cleavage site Leu-Tyr-Lys-Arg192 within
. If Kex2p cleavage after Lys-Arg192 did occur in vivo, a
/
fragment of about 15·5 kDa would have been expected; however, no such signal was detectable (see also Fig. 3b
). In addition, in vivo expression of a mutant pptox in which the Kex2p cleavage site within
(Lys-Arg192) had been destroyed in its P1 position and changed into Lys-Ala192 resulted in a normal killer phenotype indistinguishable from that of yeast transformants expressing the wild-type pptox (data not shown).
Since the endopeptidase cleavage site immediately upstream of the mature ß N terminus already resembles a classical Kex2p site (Leu-Gln-Lys-Arg245), we asked if a change in the P2 position (Leu-Gln-Gln-Arg245) prevents or weakens Kex2p cleavage activity at this site. In vivo expression of the mutated Gln-Arg245 pptox construct resulted in immune non-killers that were significantly inhibited in toxin secretion. While no detectable signal for a correctly processed /ß toxin was seen in immunoblots of cell-free culture supernatants, Western analysis of subcellular fractions enriched for endosomal and/or secretory vesicles indicated that Gln-Arg245 expressing yeasts are able to produce and secrete traces of a correctly processed 10·9 kDa ß subunit (Fig. 4
). The low amounts of ß seen within the secretory vesicle fraction of Gln-Arg245 expressing cells might be due to the residual capacity of Kex2p to partially process the mutated toxin precursor, since the same result was obtained in a yeast
yps1 mutant, while no such signal was detectable in the
kex2 mutant strain BFY113 (data not shown).
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DISCUSSION |
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Kex2p-mediated pptox processing
Sequence analysis of the K28 toxin precursor did not allow precise predictions on how both termini in are generated in vivo. The most predictable processing sites are after Leu-Glu-Glu-Arg49 (generating the
N terminus) and after Ile-Gln-Ser-Arg149 producing the
C terminus. In each case, this motif is the only basic residue in the vicinity that might mark a cleavage site for known yeast processing endoproteases. Optimal recognition sites for the Kex2p endopeptidase are Lys-Arg or Arg-Arg with preference for sites with a hydrophobic residue in the P4 position (Tao et al., 1990
; Park et al., 1994
), although it has recently been shown that the
N terminus of the K1 toxin is generated by Kex2p cleavage after Pro-Arg44 (Zhu et al., 1992
); Glu-Arg49 and Ser-Arg149, however, have not yet been reported as being recognized by Kex2p.
To shed more light onto the K28 precursor processing in yeast, we specifically destroyed/altered the predicted endopeptidase cleavage sites within the pptox wild-type sequence, expressed both wild-type and mutated pptox genes in a wild-type yeast (Kex2+ Yps1+), in a kex2 null mutant and in a
kex2
yps1 double mutant, and subsequently analysed their phenotypic effects on K28 toxicity and protein secretion. Interestingly, expression of wild-type pptox in a
kex2 mutant resulted in a cell-associated 42 kDa protein whose size corresponded to that predicted for an ER precursor after SP cleavage and addition of the three core N-glycosyl groups within
. Since the unprocessed 42 kDa protoxin was not secreted and was only detectable within the intracellular membrane fraction, it can be speculated that in a
kex2 mutant the protoxin is probably retranslocated into the cytosol and targeted for proteasomal degradation.
After in vivo expression of mutated pptox derivatives in which either of the two -flanking Kex2p sites had been destroyed (Ile-Glu-Glu-Ala49, Ile-Gln-Ser-Ala149 and the Ala49/Ala149 double mutant), immune non-killers were obtained that secreted incorrectly matured K28 processing intermediates. In case of the Ala49 single mutant and the Ala49/Ala149 double mutant,
/
- and/or
/
/
-containing pptox fragments were secreted whose N termini (Met37-Pro-Thr-Ser-Glu-Arg-Gln-Gln-Gly-Leu) corresponded exactly to the N terminus of
after SP cleavage within the ER lumen. Since no other
-containing toxin fragment was detectable in the cell-free culture supernatant of the corresponding yeast transformant and the same results were obtained after in vivo expression of a mutant Lys-Ala192 toxin derivative, it can be concluded that Kex2p endopeptidase cleavage within
(Leu-Tyr-Lys-Arg192) does not occur in vivo. In contrast, a more recent study on K1 toxin precursor processing indicated that the sequence Tyr-Val-Lys-Arg188 (which is also located within a
sequence flanking
and ß) is cleaved by Kex2p, even though the context does not fit the proposed consensus for Kex2p cleavage (Tao et al., 1990
; Park et al., 1994
). Interestingly, changing the K28 wild-type sequence Ile-Gln-Ser-Arg149 into the classical Kex2p cleavage site Ile-Gln-Lys-Arg149 did not have any effect on killer phenotype expression, and yeast cells expressing such a toxin derivative showed normal killing and were perfectly capable of secreting wild-type levels of a correctly processed
/ß heterodimeric protein toxin (data not shown). The possibility that, in the mature
-toxin, both termini are generated by the action of the monobasic endopeptidase Yps1p (Egel-Mitani et al., 1990
; Ledgerwood et al., 1996
) is highly unlikely since killer phenotype expression in a yeast
yps1 null mutant (strain HKY20-11A) was not negatively affected and secretion of a biologically active
/ß heterodimeric protein toxin resembled wild-type level (Table 1
).
Besides being responsible for the N- and C-terminal processing of , Kex2p is also involved in the N-terminal processing of the toxins ß subunit, since amino acid sequence analysis of the purified toxin identified that ß initiates after Leu-Gln-Lys-Arg245, which represents a classical Kex2p site highly predictable for endopeptidase cleavage in vivo. Expression of wild-type pptox in a
kex2 null mutant as well as in a
kex2
yps1 double mutant always resulted in non-killer yeasts that were completely blocked in toxin secretion and which accumulated a 42 kDa protoxin within the intracellular membrane fraction. Since in both mutants, activity and secreted protein were fully restored by co-transformation with YEp6-KEX2 (a KEX2 carrying 2µ vector), we conclude that K28 pptox processing indeed is Kex2p dependent.
A single disulfide bond joins the heterodimeric virus toxin and exposes the toxins ß C-terminal HDEL signal
In the mature heterodimeric protein toxin, and ß are covalently linked by a single disulfide bond. Since the
subunit possesses only one cysteine residue close to its N terminus, this cysteine residue (Cys56) must be involved in disulfide bond formation. Correspondingly, a toxin derivative in which the cysteine residue in
had been destroyed (Cys
Trp56) was biologically inactive since intermolecular disulfide bond formation was prevented. In contrast to the cytotoxic
subunit, ß has four cysteine residues, each of them being possibly involved in disulfide bond formation. We have chosen Cys340 as the most promising candidate for cysteine mutagenesis because this residue is right next to the toxins ß C-terminal HDEL signal, which has recently been shown to be absolutely required for retrograde toxin transport in a sensitive target cell (Eisfeld et al., 2000
). Once the toxin has entered a cell by endocytosis, it travels the secretion pathway in reverse (via Golgi and ER) in order to reach the yeast cell cytosol where the cytotoxic signal is transmitted into the nucleus (Schmitt & Eisfeld, 1999
). In this respect the virally encoded K28 yeast toxin resembles certain bacterial and plant toxins which likewise are able to enter and kill a eukaryotic target cell by modifying essential cellular components within the cytosol. For some of these heterodimeric protein toxins (like Pseudomonas exotoxin A and yeast K28 virus toxin) it has been shown that C-terminal K/HDEL-like sequences are responsible for intracellular targeting and retrograde transport of the toxins (Eisfeld et al., 2000
; Yoshida et al., 1991
). Correspondingly, mutations in the C-terminal ER targeting sequence dramatically reduce cytotoxicity because interaction of the toxins C terminus with the K/HDEL receptor of the target cell is prevented (Chaudhary et al., 1990
). In this respect it is also interesting to note that some of these toxins contain disulfide bonds at or near their C termini whose in vivo function is predicted to ensure access of the toxins KDEL/HDEL signal to the K/HDEL-receptor of the corresponding target cell (Pelham et al., 1992
). This situation is also true for the K28 toxin since we now show that the cysteine residue right next to the ß C terminus (Cys340) is part of the disulfide bond that covalently joins
and ß. Interestingly, yeast cells expressing the mutated Cys
Ser340 toxin derivative showed normal levels of toxin secretion, and Western analysis of cell-free culture supernatants further indicated that a heterodimeric protein is secreted that consists of a 10·5 kDa
and a 10·9 kDa ß subunit, indistinguishable from the two subunits in the Cys340 wild-type toxin. It therefore can be concluded that heterodimer formation in the mutated toxin must be catalysed by an alternative disulfide bond between Cys56 (in
) and one of the three remaining non-mutated cysteine residues in ß. Phenotypic analysis of yeasts expressing the mutated toxin identified the Cys
Ser340 toxin as being completely inactive, incapable of killing a sensitive yeast cell. Additional cell fractionation experiments on sensitive yeasts treated with the mutant Cys
Ser340 toxin indicated that the observed loss of toxicity is likely to be caused by its inability to retrograde pass a cell and to successfully reach its intracellular target. In this respect, the phenotype of a Cys
Ser340 toxin derivative exactly portrays the phenotype of a truncated toxin in which the ß C-terminal HDEL sequence had been deleted; in vivo such a toxin (ß-
HDEL) is likewise no longer capable of entering the secretion pathway of a sensitive target cell (Eisfeld et al., 2000
). We therefore postulate that in the wild-type toxin, formation of a disulfide bond between Cys56 (
) and Cys340 (ß) has the important in vivo function of ensuring accessibility of the ß C-terminal ER targeting signal to the HDEL-receptor of the target cell (Fig. 6
). It will need further experiments to actually demonstrate that disulfide bond formation controls the accessibility of the HDEL signal, and by surface plasmon resonance (BIAcore analysis) we are currently determining the in vitro binding kinetics (association and dissociation constants) between the cellular HDEL receptor Erd2p and various mutant toxins that are defective in correct disulfide bond formation.
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ACKNOWLEDGEMENTS |
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Received 10 September 2001;
revised 14 December 2001;
accepted 17 December 2001.