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A Role for the Protease-sensitive Loop Region of Shiga-like Toxin 1 in the Retrotranslocation of Its A1 Domain from the Endoplasmic Reticulum Lumen

2005; Elsevier BV; Volume: 280; Issue: 24 Linguagem: Inglês

10.1074/jbc.m414193200

1083-351X

Paul LaPointe, Xin Wei, Jean Gariépy,

Bacterial Genetics and Biotechnology

Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A1 domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A1 fragment in Saccharomyces cerevisiae. Expression of the Slt-I A1 domain (residues 1–251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1–239 represent the minimal A1 segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240–251 restores the translocation property of the A1 chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A1 domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240–251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A1 chain (residues 240–251) rather than a unique ER export signal sequence. Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A1 domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A1 fragment in Saccharomyces cerevisiae. Expression of the Slt-I A1 domain (residues 1–251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1–239 represent the minimal A1 segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240–251 restores the translocation property of the A1 chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A1 domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240–251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A1 chain (residues 240–251) rather than a unique ER export signal sequence. Shiga-like toxin I (Slt-I) 1The abbreviations used are: Slt-I, Shiga-like toxin I; DETOX, catalytically inactive Slt-I A chain; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; ERT, endoplasmic reticulum-routing toxin; PBS, phosphate-buffered saline. 1The abbreviations used are: Slt-I, Shiga-like toxin I; DETOX, catalytically inactive Slt-I A chain; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; ERT, endoplasmic reticulum-routing toxin; PBS, phosphate-buffered saline. is a member of a class of ER-routing protein toxins (ERTs) that undergo retrograde traffic to the ER of cells before relocating their enzymatic domain to the cytosol (1Yoshida T. Chen C.C. Zhang M.S. Wu H.C. Exp. Cell Res. 1991; 192: 389-395Crossref PubMed Scopus (144) Google Scholar, 2Donta S.T. Beristain S. Tomicic T.K. Infect. Immun. 1993; 61: 3282-3286Crossref PubMed Google Scholar, 3Sandvig K. Garred O. Prydz K. Kozlov J.V. Hansen S.H. van Deurs B. Nature. 1992; 358: 510-512Crossref PubMed Scopus (378) Google Scholar). Slt-I is composed of a catalytic A subunit non-covalently associated with a pentamer of B subunits responsible for binding to the glycolipid receptor, CD77 (also known as globotriaosylceramide or Gb3) (4Jacewicz M. Clausen H. Nudelman E. Donohue-Rolfe A. Keusch G.T. J. Exp. Med. 1986; 163: 1391-1404Crossref PubMed Scopus (307) Google Scholar, 5Lindberg A.A. Brown J.E. Stromberg N. Westling-Ryd M. Schultz J.E. Karlsson K.A. J. Biol. Chem. 1987; 262: 1779-1785Abstract Full Text PDF PubMed Google Scholar, 6Lingwood C.A. Law H. Richardson S. Petric M. Brunton J.L. De Grandis S. Karmali M. J. Biol. Chem. 1987; 262: 8834-8839Abstract Full Text PDF PubMed Google Scholar). Receptor binding is followed by clathrin-mediated endocytosis and retrograde transport of the toxin through the Golgi apparatus en route to the ER lumen (3Sandvig K. Garred O. Prydz K. Kozlov J.V. Hansen S.H. van Deurs B. Nature. 1992; 358: 510-512Crossref PubMed Scopus (378) Google Scholar, 7Johannes L. Goud B. Trends Cell Biol. 1998; 8: 158-162Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). While migrating through the secretory pathway, a protease-sensitive loop (residues 242–261) located in the C-terminal region of the A chain is cleaved, dividing the A chain into a B pentamer-associated A2 domain (residues 252–293) and an enzymatic A1 domain (residues 1–251) (8Garred O. van Deurs B. Sandvig K. J. Biol. Chem. 1995; 270: 10817-10821Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The A1 domain remains associated with the A2/B subunits complex by virtue of a disulfide bond between cysteines 242 and 261. This disulfide bond is ultimately reduced in the ER lumen, liberating the enzymatic A1 domain that is subsequently retrotranslocated to the cytosol. The N-glycosidase activity of the A1 fragment depurinates a single adenosine residue at position 4324 in the 28 S rRNA inhibiting protein synthesis and subsequently leading to cell death (9Endo Y. Tsurugi K. Yutsudo T. Takeda Y. Ogasawara T. Igarashi K. Eur. J. Biochem. 1988; 171: 45-50Crossref PubMed Scopus (617) Google Scholar). The mechanism by which the catalytic domain of Slt-I and other ERTs is exported from the ER lumen to the cytosol remains poorly defined. Proteolysis of the Slt-I A chain to an A1 fragment is a required event for toxicity to occur in mammalian cells (10Lea N. Lord J.M. Roberts L.M. Microbiology. 1999; 145: 999-1004Crossref PubMed Scopus (24) Google Scholar). Proteolysis exposes a hydrophobic peptide present in the A chain of Slt-I and other ERTs, an event that may facilitate its interaction with ER-resident proteins or the ER membrane (11Menikh A. Saleh M.T. Gariepy J. Boggs J.M. Biochemistry. 1997; 36: 15865-15872Crossref PubMed Scopus (45) Google Scholar, 12Saleh M.T. Ferguson J. Boggs J.M. Gariepy J. Biochemistry. 1996; 35: 9325-9334Crossref PubMed Scopus (23) Google Scholar, 13Day P.J. Pinheiro T.J. Roberts L.M. Lord J.M. Biochemistry. 2002; 41: 2836-2843Crossref PubMed Scopus (65) Google Scholar). However, studying the role of this region in retrotranslocation in isolation of other processes of retrograde traffic and processing remains challenging in the context of cell-based assays. Yeast represents an attractive model for analyzing how the enzymatic A1 domain of Slt-I is able to escape from the lumen in the ER and kill the host cell. We report the development of a suicide assay in yeast where the fully active enzymatic A1 fragment of Slt-I is rapidly targeted to the ER before it can accumulate in the cytosol. The ability of the Slt-I A1 fragment to subsequently retrotranslocate to the cytosol from the ER is lethal to the host and was the basis of a screen to define a region of the toxin involved in this relocation process. This yeast assay was used to identify a C-terminal region of the Slt-I A1 fragment involved in retrotranslocation. Plasmids and Strains—pRSATT was created by amplifying two regions of the GAL1 promoter from pRS854 and cutting them into the multiple cloning site of pRS316. The first region was created with a sense primer (STATA2, GAG AGA GAA TTC tac gct taa ctg ctc att gc) complementary to the sequence upstream of the GAL4 repeats and an antisense primer (ATATA2, GAG AGA GGA TCC GAG AGA GCA TGC gtt aat aga tca aaa atc atc gct tcg ctg) complementary to the sequence around the TATA element of the GAL1 promoter. This construct resulted in a product with an upstream EcoRI cloning site (underlined) and a downstream BamHI site (with a nested SphI site, both underlined), which was cloned into pRS316 between EcoRI and BamHI (creating an intermediate vector). The second region was created with a sense primer (SsphI, GAG AGA GCA TGC gta aat gca aaa act gca taa cca c) complementary to the TATA region of the GAL1 promoter and an antisense primer (AbamHI, GAG AGA GGA TCC ggg gtt ttt tct cct tga cg) complementary to the -1 region of the GAL1 promoter. The resulting product incorporated an upstream SphI site and a downstream BamHI site (both underlined) and was subsequently cloned into the intermediate vector containing the first fragment between SphI and BamHI. The final vector pRSATT-ER was created by cloning an ER-targeting sequence into the BamHI site of pRSATT. The sequence was created by gene synthesis from two complementary primers (fER, GAG AGA AGA TCT atg atg aag aaa aac aat gcg tta gca cta gcg ctt gcc ctt gcg cta gca ctg gct ttg gcc ctg gcc and rER, GAG AGA GGA TCC cgc gtt ggc ggt gcc tag cgc aag tgc aag cgc tag tgc taa cgc taa ggc cag ggc caa agc cag) with BglII and BamHI sites, respectively (both underlined). The primers were annealed and extended in a PCR reaction and subcloned into pBLUESCRIPT. The insert was then cut out with BglII and BamHI and cloned into the BamHI site of pRSATT. DNA cloning was carried out in the Escherichia coli strain DH5 [F-.80dlacZ M15 (lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rK-, mk+) supE44- thi-1 gyrA96 relA1). The wild-type yeast strain W303a was provided by James Friesen (Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada). RSY1293 and 1295 strains were obtained from Randy Schekman (Department of Cell and Developmental Biology, University of California at Berkeley). Construction of Yeast Expression Vectors Encoding Slt-I A—Cassettes encoding portions of the Slt-I A chain coding sequence were cloned into pRSATT and pRSATT-ER between BamHI and SacI. These cassettes were all generated by PCR using the sense primer P1 in combination with the antisense primers (D1 through D13). The primers and corresponding products are shown in Table I. The overexpression plasmids (for the immunoprecipitation experiments) were created by cloning the wild-type Gal1 promoter into pRS416 between EcoRI and BamHI (creating pRS416Gal). The BglII-BamHI insert (coding for the ER-targeting sequence used to make pRSATT-ER) was then cloned into the BamHI site of pRS416Gal creating pRS416GalER. Cassettes coding for N-terminally myc-tagged and catalytically inactive (DT) A1 variants were generated by PCR using the sense primer P2 in combination with D1 and D6 (1–251 and 1–239 fragments, respectively) and cloned into pRS416Gal and pRS416GalER between BamHI and SacI. Thirty cycles of amplification (denature 94 °C, 30 s; anneal 58 °C, 30 s; extension 72 °C, 1 min) were carried out with a PerkinElmer Life Sciences thermal cycler and Pwo polymerase (Roche Applied Science). The PCR products and vectors were cut with BamHI and SacI (Roche Applied Science) and cloned using T4 DNA ligase (New England Biolabs).Table IPrimers used in this study to construct expression vectorsProductSenseP1GAG AGA GGA TCC ATG aag gaa ttt acc tta gacP2GAG AGA GGA TCC ATG gag caa aag ctc att tct gaa gag gac ttg aag gaa ttt acc tta gacmyc-taggedAntisenseD1GAG AGA GAG CTC TCA CTA tct ggc aac tcg cga tgcAC SacI 251 TAG TGAD2GAG AGA GAG CTC TCA CTA atg gtg atg aca att cag tat taa tgc cac gct tccAC SacI 245 TAG TGAD3GAG AGA GAG CTC TCA CTA aca att cag tat taa tgc cac gct tccAC SacI 242 TAG TGAD4GAG AGA GAG CTC TCA CTA att cag tat taa tgc cac gct tccAC SacI 241 TAG TGAD5GAG AGA GAG CTC TCA CTA cag tat taa tgc cac gct tccAC SacI 240 TAG TGAD6GAG AGA GAG CTC TCA CTA tat taa tgc cac gct tcc cagAC SacI 239 TAG TGAD7GAG AGA GAG CTC TCA CTA taa tgc cac gct tcc cagAC SacI 238 TAG TGAD8GAG AGA GAG CTC TCA CTA agc tat taa tgc cac gct tcc cag aat tgcAC SacI L240A 240D9GAG AGA GAG CTC TCA CTA att tat taa tgc cac gct tcc cag aat tgcAC SacI L240N 240D10GAG AGA GAG CTC TCA CTA acg tat taa tgc cac gct tcc cag aat tgcAC SacI L240R 240D11GAG AGA GAG CTC TCA CTA gtc tat taa tgc cac gct tcc cag aat tgcAC SacI L240D 240D12GAG AGA GAG CTC TCA CTA tct ggc aac tcg cga tgc atg gtg atg aca att agc tat taa tgc cac gct tcc cag aat tgcAC SacI L240A 251D13GAG AGA GAG CTC TCA CTA tct ggc aac tcg cga tgc atg gtg atg aca att att tat taa tgc cac gct tcc cag aat tgcAC SacI L240N 251D14GAG AGA GAG CTC TCA CTA tct ggc aac tcg cga tgc atg gtg atg aca att acg tat taa tgc cac gct tcc cag aat tgcAC SacI L240R 251D15GAG AGA GAG CTC TCA CTA tct ggc aac tcg cga tgc atg gtg atg aca att gtc tat taa tgc cac gct tcc cag aat tgcAC SacI L240D 251 Open table in a new tab mRNA Isolation, Reverse Transcription-PCR, and Amplification— Total RNA was recovered from 107 yeast cells grown at 30 °C in SC-uracil medium made with 2% galactose. Cells were harvested in mid-log phase and RNA purified with the Qiagen RNeasy mini kit. RT-PCR was performed with the 1st Strand cDNA synthesis kit (Amersham Biosciences) according to manufacturer's instructions. PCR of cDNA was performed with primers P and D6 for 40 cycles. Steady-state Determination of Slt-I A1 Levels in the Cytosol and ER—Twenty-five (25) ml cultures of W303a yeast cells transformed with pRS416, pRS416Gal mycDT1–251, pRS416GalER mycDT1–251, pRS416Gal mycDT1–239, and pRS416GalER mycDT1–239 were grown to an A600 of 0.8. The cells were washed once in buffer 1 (100 mm Tris, pH 9.4, 10 mm dithiothreitol) and then resuspended in 1 ml of spheroplasting buffer (20 mm Tris pH 7.5, 1 m sorbitol, 2 μg/ml zymolyase 100-T, in SC-uracil 2% galactose broth) and incubated for 1 h at 37 °C. Spheroplasts were pelleted and resuspended in 1 ml of lysis buffer (20 mm HEPES, pH 6.8, 50 mm potassium acetate, 200 mm sorbitol, 2 mm EDTA, with protease inhibitors). Spheroplasts were lysed in a Dounce homogenizer on ice. The lysate was centrifuged for 5 min at 500 × g to remove unbroken cells and nuclei. The supernatant was centrifuged for 1 h at 100,000 × g in a TLA-100 ultracentrifuge to pellet ER membranes. The remaining cytosolic components in the supernatant were precipitated with trichloroacetic acid, washed in acetone and resuspended in 1 ml of RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) prior to immunoprecipitation with a mouse anti-myc monoclonal antibody at 70 μg/ml (9E10, kind gift from Bill Balch, The Scripps Research Institute). Briefly, the ER membrane pellet was washed once with lysis buffer and resuspended in 1 ml RIPA buffer. Immune complexes formed overnight at 4 °C were captured on protein-G-Sepharose beads for 30 min at 4 °C. The beads were washed once with PBS and then boiled in sample buffer for SDS-PAGE. A1 constructs were detected by Western blot analysis using rabbit anti-Slt-I A chain polyclonal antibodies (Molecular Templates Inc., Toronto). Yeast Transformation and Growth Properties (Spot Assay)—Yeast cells were transformed according to the lithium-acetate method (14Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Five-ml overnight cultures were grown at 30 °C of selective media supplemented with 2% glucose. These cultures were then pelleted and washed twice with PBS and resuspended in 1 ml of PBS. Samples were then diluted to an A600 of 1.0 and serially diluted (10×). Each dilution was then spotted (10 μl) on SD-Ura plates supplemented with 2% galactose. The plates were incubated at 24, 30, or 37 °C for 72 h. Recombinant His8-tagged Protein Expression, Purification, and Rabbit Reticulocyte Lysate Assay—Constructs coding for the Slt-I A1 fragments 1–238, 1–239, and 1–251 were cloned into the bacterial expression vector pET22b (Novagen) and the fragments expressed as N-terminal His-tag fusion proteins. Constructs coding for Slt-I holotoxins (wild-type and DETOX) were cloned into the pECHE10a vector (Molecular Templates Inc., Toronto, Ontario, Canada), expressed with a His8-tag at the N terminus of their A chain and purified using nickel-nitrilotriacetic acid resin (Qiagen). The enzymatic activity of these molecules was tested in a rabbit reticulocyte lysate assay (TNT assay kit, Promega) using the expression of luciferase as a measure of protein synthesis. The loss of relative light units is related to the ribosome-inactivating activity of Slt-I A chains. Cytotoxicity Assay on Vero Cells—The toxicity of wild-type Slt-I and the variant Slt-I (L240D) toward Vero cells were measured using the sulforhodamine B dye binding assay (15Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1107-1112Crossref PubMed Scopus (8824) Google Scholar). Fifty-thousand Vero cells in 200 μl of α-minimal essential medium were cultured in wells of 96-well plates and exposed to a range of toxin concentrations prepared in PBS for a 1-h period. The toxin-containing solutions were subsequently diluted with the appropriate medium containing fetal calf serum, and the treated cells were cultured for another 48 h. The medium was removed and the remaining adherent cells were fixed with ice-cold 10% trichloroacetic acid, air-dried, and stained with 0.4% sulforhodamine B (Molecular Probes, Eugene, OR) dissolved in 1% (v/v) acetic acid in water. The excess dye was washed away, and the remaining bound sulforhodamine B dye was extracted from the cells with 10 mm Tris base. The absorbance of the dye was read at 540 nm using a plate reader. Each point in the cytotoxicity curves represents the average of experiments performed in triplicate. Circular Dichroism Experiments—CD spectra were recorded on an Aviv 62A DS circular dichroism spectrometer using a 0.5-cm path length rectangular cuvette with a 2-ml sample volume. Protein samples (16.9 μm) of N-terminal His-tagged Slt-I A1 fragments 1–238, 1–239, and 1–251 were prepared in sample buffer (25 mm sodium phosphate, pH 7.0, and 100 mm NaCl). Wavelength scans were recorded from 300 to 195 nm, with a 1-nm spectral bandwidth (1 nm between points) and an averaging time of 8 s. Triplicate spectra were recorded for each protein sample. Expression of the Slt-I A1 Fragment in Yeast—Two yeast expression vectors were constructed to study the retrotranslocation of the A1 fragment of Shiga-like toxin 1 (Fig. 1A). The first vector, termed pRSATT, was constructed to express Slt-I A1 chain variants directly into the cytosol under the control of an attenuated Gal1 promoter (16Selleck S.B. Majors J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5399-5403Crossref PubMed Scopus (21) Google Scholar). The second vector, pRSATT-ER, was designed such that Slt-I A1 variants would be expressed with an N-terminal ER-targeting sequence (17Hatsuzawa K. Tagaya M. Mizushima S. J. Biochem. (Tokyo). 1997; 121: 270-277Crossref PubMed Scopus (48) Google Scholar) to ensure the concomitant synthesis and translocation of the nascent A1 chains into the ER lumen. Folded A1 mutant chains unable to retrotranslocate from the ER (or another compartment of the secretory pathway) back to the cytosol would result in a survival phenotype. A comparison of toxicity levels observed for cloned Slt-I A1 variants expressed from pRSATT and pRSATT-ER was the basis of a method to delimit region(s) of the A1 chain involved in retrotranslocation (Fig. 1B). Identification of a Retrotranslocative C-terminal Peptide in the Slt-I A Chain—We hypothesized that distinct peptide domains mediated cytotoxicity and retrotranslocation and that a minimum cytotoxic fragment of the Slt-I A1 fragment would not retain its ability to escape from the ER lumen. A series of cassettes coding for the wild-type Slt-I A1 fragment (residues 1–251) and C-terminally truncated variants were created and cloned into pRSATT and pRSATT-ER. The expression of the 1–251 Slt-I A1 fragment from pRSATT was lethal to the host (Fig. 2A; fragment 1–251). Slt-I A1 fragments truncated at their C terminus also remained lethal to the host until residue 239 was deleted (Fig. 2A; fragments 1–238 and 1–239). The viability of yeast cells transformed with the pRSATT vector coding for residues 1–238 was indistinguishable from that observed in yeast transformed with the empty vector (pRSATT only) and in yeast expressing a catalytically inactive E167A/R170A double mutant termed DETOX (Fig. 2A). Wild-type Slt-I holotoxin and its detoxified variant as well as Slt-I A1 fragments coding for either residues 1–238, 1–239, or the entire A chain were then separately expressed in bacteria, purified, and subsequently tested to confirm their ability to inactivate ribosomes in vitro (N-glycosidase activity) in a rabbit reticulocyte lysate (TNT) assay (Fig. 2C). As expected, the enzymatic activities of the A chain and of the 1–239 fragment were comparable with that of the wild-type Slt-I toxin, while the 1–238 fragment and the DETOX form of the toxin had no detectable catalytic activity (Fig. 2C). Genes coding for Slt-I A1 1–251 and 1–239 were then cloned into pRSATT-ER to determine whether the deleted C-terminal region (residues 240–251) was essential for routing the A1 fragment from the ER lumen to the cytosol. Both Slt-I A1 1–251 and 1–239 were equally toxic when expressed into the cytosol of yeast cells (Fig. 2B), while only the 1–251 construct remained toxic to cells when expressed with an N-terminal signal sequence (Fig. 2B, fragment ER 1–251). A survival phenotype was observed from the ER-routed A1 1–239 variant implying that the truncated protein was unable to relocate back to the cytosol from the ER lumen or any other compartments along the secretory pathway or was simply secreted by the yeast cells (Fig. 2B; fragment ER 1–239). In view of the extreme toxicity of ribosome-inactivating proteins (18Yamaizumi M. Mekada E. Uchida T. Okada Y. Cell. 1978; 15: 245-250Abstract Full Text PDF PubMed Scopus (587) Google Scholar) such as Slt-I, yeast vectors harboring a strongly attenuated Gal1 promoter were used in this study to minimize the expression of Slt-I A1 and thus avoid overwhelming the ER import pathway. As a result of this experimental constraint, cytotoxic A1 variants could not be detected by conventional biochemical techniques (Western blot, immunoprecipitation, microscopy) when expressed from the attenuated promoter. Three sets of experiments were then performed to confirm that the Slt-I A1 fragment is translocated into the ER lumen and that the survival of yeast cells expressing the ER-targeted 1–239 fragment was not due to the lack of toxin production. First, the expression of messenger RNAs was confirmed using RT PCR for all A1 mutants targeted to the endoplasmic reticulum and leading to a survival phenotype in yeast (Fig. 3A). Second, catalytically inactive forms (E167A/R170A) of the A1 chain variants 1–251 and 1–239 were constructed and overexpressed in yeast cells to monitor their distribution within cellular compartments. More specifically, myc-tagged versions of the 1–251 and 1–239 fragments were cloned into a high copy number vector (pRS416) and expressed under the control of the wild-type Gal1 promoter, with and without the ER signal sequence. W303a yeast cells were then transformed with these vectors and grown in galactose to induce the expression of the toxin variants. The yeast cells were fractionated into cytosolic and ER fractions, and the myc-tagged protein was immunoprecipitated from each fraction. Immunoprecipitated material was then separated by SDS-PAGE and Western blots were performed using anti-Slt-I A chain polyclonal antibodies. As expected, 1–251 and 1–239 toxin fragments were recovered from the cytosol but not from ER fractions of yeast cells expressing the 1–251 and 1–239 toxin fragments lacking the ER signal sequence (∼29-kDa band; Fig. 3B). Similarly, toxin was only recovered from the cytosol of yeast cells expressing the 1–251 fragment fused to the ER signal sequence (∼33-kDa band; Fig. 3B). In this case, a larger molecular mass band (∼33 kDa band; 1–251 fragment with ER signal sequence) is observed in the cytosol as expected from the cytoplasmic overexpression of this construct as well as a dominant cleaved A1 chain band (∼29 kDa band) corresponding to the mass of fragment 1–251 lacking the ER signal sequence. These results suggest that the A1 chain has been directed into the ER and then rerouted to the cytosol after removal of the signal sequence. Finally, the fragment expressing residues 1–239 fused to the signal-cleaved ER signal sequence was predominantly found in the ER fraction of yeast cells (∼29-kDa band) suggesting that it accumulated there after translocation and signal cleavage. Predictably, some of the overexpressed ER signal-containing version of the 1–239 fragment (∼33 kDa band) was also observed in its unprocessed form the cytosolic fraction. Last, a genetic approach was devised to confirm that the survival phenotype of yeast expressing the ER-targeted 1–239 fragment was due to ER import and not due to a lack of enzymatic activity. Our strategy was based on the use of a cold-sensitive yeast strain, which expresses a mutant form of Sec61p that conditionally limits ER import. The strain RSY1295 is non-viable at 17 °C but propagates in the temperature range from 23 to 37 °C where the disruption in ER import is not so severe as to prevent cell growth (19Pilon M. Schekman R. Romisch K. EMBO J. 1997; 16: 4540-4548Crossref PubMed Scopus (347) Google Scholar, 20Pilon M. Romisch K. Quach D. Schekman R. Mol. Biol. Cell. 1998; 9: 3455-3473Crossref PubMed Scopus (52) Google Scholar). Transforming RSY1295 with the vector expressing ER-targeted 1–239 (ER 1–239; Fig. 3C) and growing this particular strain at a temperature causing a partial blockage of ER import should lead to a reduction in yeast viability due to the accumulation of newly synthesized toxic 1–239 fragments directly into the cytosol. At the permissive temperature for ER import (30 °C), RSY1295 displayed the expected sensitivity to cytosolically expressed DETOX, 1–251 and 1–239, and ER-targeted 1–251 and 1–239 (Fig. 3C; DETOX, 1–251, 1–239, ER 1–251, and ER 1–239) as was seen for the isogenic wild-type RSY1293 strain transformed with the same vectors. However, at 37 °C, RSY1295 was 2 orders of magnitude more sensitive to the ER-targeted 1–239 than RSY1293 (Fig. 3C, ER 1–239). The same effect was observed, albeit to a lesser extent, at 24 °C (Fig. 3C) indicating that the toxic A1 fragment 1–239 with the N-terminal ER-targeting sequence was produced but poorly shuttled to the ER lumen, thereby resulting in ribosome inactivation. Similar results were obtained for all non-retrotranslocative A1 mutants expressed in the cold-sensitive RSY1295 yeast strain (data not shown). Taken together, these results suggest that A1 chains fused to the ER signal sequence are produced as enzymatically active molecules and targeted to the ER lumen. Furthermore, the data indicate that the 1–251 fragment of the Slt-I A chain efficiently retrotranslocates from the ER, while the 1–239 fragment accumulates there. Delimiting the Retrotranslocative Peptide of the Slt-I A1 Fragment—Progressively longer versions of the Slt-I A1 fragment were cloned into pRSATT-ER and expressed in yeast to determine the minimal peptide segment within the region 240–251 necessary to facilitate the retrotranslocation of the A1 chain from the ER lumen to the cytosol. The addition of Leu240 to the C terminus of 1–239 had a dramatic effect on retrotranslocation, restoring toxicity to nearly that of the wild type A1 domain (Fig. 4A, ER 1–240 and ER 1–251). The additions of Asn241 and Cys242 appear to partly mask the retrotranslocative potential of Leu240, while the addition of the three consecutive histidines from residues 243 to 245 restored retrotranslocation to the same level observed for 1–251 (Fig. 4A, ER 1–241, ER 1–242, and ER 1–245). The toxicity of all lengths tested was indistinguishable from wild type when expressed from pRSATT (data not shown). Substitution of Leucine for Aspartic Acid at Position 240 in the 1–240 and 1–251 Slt-I A1 Fragment Results in a Reduction in Retrotranslocative Potential—The addition of Leu240 to the 1–239 fragment of the Slt-I A chain restored most of its retrotranslocative potential. To determine whether this effect was due to the presence of a specific amino acid at position 240 or simply a chain length effect, leucine 240 was replaced in the 1–240 Slt-I A1 fragment with alanine, asparagine, arginine, and aspartic acid and tested for cytotoxicity when expressed and routed to the ER using the pRSATT-ER vector. The toxicity of these ER-targeted variants was reduced (Fig. 4B) in relation to the ER-targeted 1–240 and 1–251 fragments (Fig. 4B). Full-length Slt-I A1 mutants (1–251) were then constructed to determine whether mutations at position 240 were blocking retrotranslocation specifically because of their location at the C terminus of the peptide. Leucine at position 240 was replaced either with alanine,