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

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 the first fragment between SphI and The final vector was created by cloning an ER-targeting sequence into the BamHI site of The sequence was created by synthesis from two complementary GAG AGA AGA aaa aat gca gca ctg gct ctg and GAG AGA GGA TCC gtt taa taa with and BamHI (both The and in a and into The was with and BamHI and cloned into the BamHI site of cloning was in the The yeast was by and of of and from of Cell and of at of Yeast Expression Slt-I of the Slt-I A chain sequence cloned into and between BamHI and by the sense primer in with the antisense through The and in The the created by cloning the promoter into between EcoRI and BamHI (creating The for the ER-targeting sequence used to was cloned into the BamHI site of for and catalytically inactive A1 variants by the sense primer in with and and 1–239 and cloned into and between BamHI and of with a and The and with BamHI and and cloned used in this study to construct expression AGA GGA TCC ttt AGA GGA TCC ctc att tct ttt AGA GAG tct tcg AGA GAG att taa gct AGA GAG att taa gct 242 AGA GAG att taa gct AGA GAG taa gct AGA GAG taa gct AGA GAG taa gct AGA GAG taa gct aat AGA GAG att taa gct aat AGA GAG taa gct aat AGA GAG taa gct aat AGA GAG tct tcg att taa gct aat AGA GAG tct tcg att att taa gct aat AGA GAG tct tcg att taa gct aat AGA GAG tct tcg att taa gct aat in a and was from yeast cells at in with in and with the was with the synthesis to of was with and for of Slt-I A1 in the and of yeast cells with and to an of The cells in and in of in and for at and in of with in a on The was for at to cells and The was for at in a to ER The in the with in and in of to with a at from The the ER membrane was with and in at on for at The with and in for A1 by A chain Yeast and cells to the H. Y. K. A. J. PubMed Google Scholar). at of with and with and in of to an of and was on with The at or for and for the Slt-I A1 and cloned into the expression vector and the as for Slt-I and cloned into the vector with a at the terminus of their A chain and The enzymatic activity of was in a assay assay the expression of as a of protein The of is to the ribosome-inactivating activity of Slt-I A on Vero toxicity of Slt-I and the Slt-I toward Vero cells the B binding assay A. J. H. S. J. PubMed Scopus Google Scholar). Vero cells in of in of and to a of toxin in for a The subsequently with the and the cells for The was and the cells with and with B in in The was and the B was from the cells with The of the was at a in the represents the of in on an a with a of Slt-I A1 and in and from to with a between and an of for protein Expression of the Slt-I A1 in yeast expression to study the retrotranslocation of the A1 fragment of Shiga-like toxin The first was to Slt-I A1 chain variants into the cytosol the of an promoter J. S. A. 1988; PubMed Scopus Google Scholar). The second was such that Slt-I A1 variants with an ER-targeting sequence K. M. S. J. Biochem. 1997; PubMed Scopus Google to the synthesis and translocation of the A1 into the ER A1 to retrotranslocate from the ER compartment of the secretory to the cytosol in a A of toxicity for cloned Slt-I A1 variants from and was the basis of a to of the A1 chain involved in retrotranslocation of a C-terminal in the Slt-I A that peptide and retrotranslocation and that a cytotoxic fragment of the Slt-I A1 fragment not its ability to escape from the ER A of for the Slt-I A1 fragment (residues 1–251) and variants created and cloned into and The expression of the Slt-I A1 fragment from was lethal to the host fragment Slt-I A1 at their C terminus lethal to the host residue was and The of yeast cells with the vector for residues was from that in yeast with the vector and in yeast a catalytically inactive Slt-I and its as as Slt-I A1 for residues or the A chain in and subsequently to their ability to in in a assay the enzymatic of the A chain and of the 1–239 fragment with that of the Slt-I the fragment and the of the toxin catalytic activity for Slt-I A1 and 1–239 cloned into to the C-terminal region (residues 240–251) was for the A1 fragment from the ER lumen to the cytosol. Slt-I A1 and 1–239 when into the cytosol of yeast cells the construct to cells when with an signal sequence fragment ER A was from the A1 1–239 that the protein was to to the cytosol from the ER lumen or other the secretory or was by the yeast cells fragment ER of the toxicity of ribosome-inactivating proteins M. E. T. Y. Full Text PDF PubMed Scopus Google such as Slt-I, yeast a promoter used in this study to the expression of Slt-I A1 and the ER a of this cytotoxic A1 variants not by when from the promoter. of to that the Slt-I A1 fragment is into the ER lumen and that the of yeast cells the 1–239 fragment was not to the of toxin the expression of was for A1 targeted to the endoplasmic reticulum and leading to a in yeast catalytically inactive of the A1 chain variants and 1–239 and in yeast cells to their within of the and 1–239 cloned into a vector and the of the with and without the ER signal sequence. yeast cells with and in to the expression of the toxin The yeast cells into and ER and the protein was from was by and A chain and 1–239 toxin from the cytosol not from ER of yeast cells the and 1–239 toxin lacking the ER signal sequence toxin was from the cytosol of yeast cells the fragment to the ER signal sequence this a fragment with ER signal is in the cytosol as from the cytoplasmic of this construct as as a A1 chain to the of fragment lacking the ER signal sequence. results suggest that the A1 chain directed into the ER and to the cytosol of the signal sequence. the fragment residues 1–239 to the ER signal sequence was in the ER of yeast cells that it translocation and signal of the ER of the 1–239 fragment was in its the a was to that the of yeast the 1–239 fragment was to ER and not to a of enzymatic activity. was based on the of a yeast which a of that ER The is at in the from to where the in ER is not as to cell M. K. J. 1997; PubMed Scopus Google Scholar, M. K. Biol. 1998; PubMed Scopus Google Scholar). with the vector 1–239 and this at a a of ER to a in yeast to the of 1–239 into the cytosol. the for ER the to DETOX, and and and 1–239 DETOX, ER and ER as was for the with the However, at was of more to the 1–239 than ER The effect was to a at that the A1 fragment 1–239 with the ER-targeting sequence was poorly to the ER lumen, resulting in results for A1 in the yeast not results suggest that A1 to the ER signal sequence as active and targeted to the ER the that the fragment of the Slt-I A chain efficiently from the ER, the 1–239 fragment accumulates the of the Slt-I A1 of the Slt-I A1 fragment cloned into and in yeast to the minimal peptide segment within the region 240–251 to facilitate the retrotranslocation of the A1 chain from the ER lumen to the cytosol. The addition of to the C terminus of 1–239 a effect on toxicity to that of the A1 domain ER and ER The of and to the of the addition of the from residues to retrotranslocation to the for ER ER and ER The toxicity of was from when from not of for at in the and Slt-I A1 in a in addition of to the 1–239 fragment of the Slt-I A chain of its To this effect was to the of a at position or a chain was in the Slt-I A1 fragment with and and for when and to the ER the The toxicity of variants was reduced in to the and Slt-I A1 to mutations at position retrotranslocation of their at the C terminus of the at position was with or in the context of the Slt-I A1 and the for toxicity when from The retrotranslocation by mutations at position in the Slt-I A1 fragment when residues to this chain that single not to the retrotranslocation event in the context of the full A1 Yeast Slt-I A1 variants at position from the vector the as yeast Slt-I A1 that of the created full enzymatic activity not the of a single in retrotranslocation was in an assay the Slt-I holotoxin. and Slt-I and their toward cells to the was toward mammalian cells. both the and toxicity toward cells 1–239 and Slt-I A1 in ER lumen proteins and that the and structural of proteins J.L. 35: PubMed Scopus Google Scholar, Cell Biol. 2002; PubMed Scopus Google Scholar). proteins subsequently to the cytosol for M. K. J. 1997; PubMed Scopus Google Scholar, A. M. 1996; PubMed Scopus Google Scholar). of is by cells to proteins and to present a complex of peptide to a of complex class I in the ER lumen M.T. J. 1995; Google Scholar, M. J. 2002; PubMed Scopus Google Scholar). ER-resident can a of altered or proteins that may as misfolded Cell Biol. 2002; PubMed Scopus Google Scholar). than the of unique peptide The Slt-I A1 domain as as other toxins may such of toxins in common a domain in sequence to residues the of residues the ER export of fragment Slt-I A1 to the that the first of Slt-I A1 not a misfolded in to the Slt-I A1 Slt-I A1 1–239 and enzymatic activity and not significantly in of their To that do in and the of Slt-I A1 A1 chain; and from to that their the of catalytic activity the of retrotranslocation can associated with a The addition of residues 240–251 may the ER export event through the of a misfolded C-terminal This of recognition not on the of a unique peptide motif and the minimal of single mutations within residues 240–251 of the peptide such as and by eukaryotic cells and migrate in a retrograde the ER catalytic domain subsequently escape from this compartment and their The A1 chain of Slt-I represents such catalytic domain and was in the present study to identify a C-terminal region a role in its retrotranslocation to the cytosol. the Slt-I A1 chain residues of Shiga-like toxin A a hydrophobic segment within its C-terminal region (residues a region to involved in its ER export mechanism M.T. Ferguson J. Boggs J.M. Gariepy J. Biochemistry. 1996; 35: 9325-9334Crossref PubMed Scopus (23) Google Scholar). hydrophobic in the A chain of and Shiga-like toxins and in other ribosome-inactivating proteins such as M.T. Ferguson J. Boggs J.M. Gariepy J. Biochemistry. 1996; 35: 9325-9334Crossref PubMed Scopus (23) Google and Lord J.M. Roberts L.M. Eur. J. Biochem. 1995; PubMed Scopus Google To define a role for this region of the A1 domain in S. cells with a suicide vector that the cytotoxic A1 domain in the cytosol of yeast cells or was to the ER This other associated with the of toxins and associated with this final translocation the expression of construct to the or of this domain in the cytosol of yeast resulting in the of and in cell death The C terminus of the A1 chain was subsequently to both the minimal catalytic domain and the domain of the A1 domain of study revealed that the first of the A1 chain the minimal region displaying full catalytic activity when in the cytosol of yeast cells. the fragment an ER-targeting sequence the ability to migrate from the ER compartment to the and was not to yeast cells and The of the chain within the context of the A1 chain subsequently demonstrated that the addition of or the segment 240–251 a similar effect in of the ability of the A1 chain to the ER lumen and kill yeast cells. A of single mutations at position revealed that the of at this site was not to the retrotranslocation event in the of the C-terminal peptide within the of the A1 chain (residues 1–251) This was when the cytotoxic activity of the toxin and a a toxicity toward Vero cells results suggest that the of the translocation mechanism is not based on the recognition of a peptide sequence rather on a peptide element, the of a misfolded or hydrophobic sequence. This is by the that the sequence of toxins not the of a peptide motif similar to residues 240–251 of Shiga-like toxin of the of the that residues of its A chain of its hydrophobic in in the Kozlov Biol. PubMed Scopus Google Scholar). of the of residues in the A1 fragment that to an the A2 and B subunit such as in yeast the The 1–239 A1 fragment (with hydrophobic residues in the C-terminal may as or in the secretory and not a for ER The addition of hydrophobic may the A1 fragment it a for the of the cell. The of with a residue such as may the of this region of the A1 fragment, a event that is in the context of the A1 fragment in yeast The that the effect in the context of the Slt-I is not this residue is not in the of the A2 fragment and B The for A1 and that a similar not The of residues 240–251 in A1 fragment to in a structural residues This and the that both A1 1–239 and active suggest that the peptide element altered or misfolded peptide that the of a hydrophobic in the A1 fragment is localized to the region for this from two the context of the Shiga-like toxin the A chain is by The site (8Garred O. van Deurs B. Sandvig K. J. Biol. Chem. 1995; 270: 10817-10821Abstract Full Text Full Text PDF PubMed Scopus (170) Google is in the A chain protease-sensitive loop (residues a region by a single disulfide bond to at or this site or at the A chain with other demonstrated that the processing of the A chain to an A1 fragment the toxicity of the toxin not alter the catalytic activity of A chain in a cell O. E. S. van Deurs B. Kozlov J.V. Sandvig K. Exp. Cell Res. 1995; PubMed Scopus Google Scholar, Roberts L.M. 1993; PubMed Scopus Google Scholar). Since the A1 fragment is from the A2 and B subunits in the ER lumen as the disulfide bond between and is the results suggest that it is at the of the retrotranslocation that ER-resident proteins may the A1 with toxin demonstrated that the of its protease-sensitive loop is required for the interaction of its A chain fragment with the ER-resident protein disulfide B. Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Cell Biol. 2002; PubMed Scopus Google Scholar). disulfide is a which a of misfolded proteins and their by the of their disulfide A. M. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proteins do not represent protein disulfide in with proteins that the mechanism of recognition is based on misfolded peptide B. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that a of ERTs the cytosol by the B. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). A misfolded peptide element in the context of their ERT, through processing as a universal mechanism leading to the recognition of by and ER-resident proteins to the can a retrotranslocation model where the of the protease-sensitive loop within the A chain of Slt-I results in the of a peptide element located at the C terminus of the A1 This peptide residues 240–251 of the A1 fragment a event leading to an interaction with ER-resident proteins involved in the recognition and retrotranslocation of misfolded proteins the that the C terminus of the A1 fragment of a region by an ER from an interaction act to the A1 fragment in the ER for a of for retrotranslocation to The of this interaction may the A1 chain to migrate to other of the secretory it for the ER retrotranslocation may that the Slt-I the ER through the interaction of its B subunits with the glycolipid (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). The A1 chain is subsequently from the B pentamer in the ER The ER-targeting is not associated with the A1 chain We a of the 1–239 fragment in the cytosol of yeast cells the 1–239 fragment This is with the that the 1–239 fragment can retrotranslocate to the ER may not long in that compartment for an retrotranslocation event to We and as as the of their for yeast and

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.