Li, Shuyu, Spooner, Robert A. , Hampton, Randolph Y., Lord, Mike (J. Mike) and Roberts, Lynne M. . (2012) Cytosolic entry of Shiga-like toxin A chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p. PLoS ONE, Vol.7 (No.7). e41119. ISSN 1932-6203

Preview

Text WRAP_Li_ournal.pone.0041119 (1).pdf - Published Version Download (2456Kb) | Preview

Abstract

Background Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. Methodology and Principal Findings Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors. Conclusions and Significance The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Escherichia coli -- Physiology, Bacterial toxins, Endoplasmic reticulum
Journal or Publication Title:	PLoS ONE
Publisher:	PLOS
ISSN:	1932-6203
Date:	2012
Volume:	Vol.7
Number:	No.7
Page Range:	e41119
Identification Number:	10.1371/journal.pone.0041119
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Funder:	Wellcome Trust (London, England)
Grant number:	080566/Z/06/Z (WT)
References:	1. Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126: 361– 373. 2. Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126: 349– 359. 3. Bays NW, Wilhovsky SK, Goradia A, Hodgkiss-Harlow K, Hampton RY (2001) HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol Biol Cell 12: 4114–4128. 4. Ye Y, Meyer HH, Rapoport TA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414: 652–656. 5. Jarosch E, Taxis C, Volkwein C, Bordallo J, Finley D, et al. (2002) Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat Cell Biol 4: 134–139. 6. Rabinovich E, Kerem A, Frohlich KU, Diamant N, Bar-Nun S (2002) AAAATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol 22: 626–634. 7. Wang Q, Li L, Ye Y (2006) Regulation of retrotranslocation by p97-associated deubiquitinating enzyme ataxin-3. J Cell Biol 174: 963–971. 8. Zhong X, Pittman RN (2006) Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates. Human Mol Genet 15: 2409–2420. 9. Elkabetz Y, Shapira I, Rabinovich E, Bar-Nun S (2004) Distinct steps in dislocation of luminal endoplasmic reticulum-associated degradation substrates: roles of endoplamic reticulum-bound p97/Cdc48p and proteasome. J Biol Chem 279: 3980–3989. 10. Lipson C, Alalouf G, Bajorek M, Rabinovich E, Atir-Lande A, et al. (2008) A proteasomal ATPase contributes to dislocation of endoplasmic reticulumassociated degradation (ERAD) substrates. J Biol Chem 283: 7166–7175. 11. Vembar SS, Brodsky JL (2008) One step at a time: endoplasmic reticulumassociated degradation. Nat Rev Mol Cell Biol 9: 944–957. 12. Majoul I, Ferrari D, Soling HD (1997) Reduction of protein disulfide bonds in an oxidizing environment. The disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum. FEBS Lett 401: 104–108. 13. Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, et al. (2004) Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase. Biochem Pharmacol 67: 1721– 1731. 14. Spooner RA, Watson PD, Marsden CJ, Smith DC, Moore KA, et al. (2004) Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum. Biochem J 383: 285–293. 15. Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, et al. (2009) Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C. J Biol Chem 284: 10232–10242. 16. Tsai B, Rodighiero C, Lencer WI, Rapoport TA (2001) Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104: 937–948. 17. Pande AH, Scaglione P, Taylor M, Nemec KN, Tuthill S, et al. (2007) Conformational instability of the cholera toxin A1 polypeptide. J Mol Biol 374: 1114–1128. 18. Spooner RA, Lord JM (2011) How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum. Curr Top Microbiol Immunol. 19. Spooner RA, Hart PJ, Cook JP, Pietroni P, Rogon C, et al. (2008) Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum. Proc Natl Acad Sci U S A 105: 17408–17413. 20. TaylorM, Navarro-Garcia F, Huerta J, Burress H, Massey S, et al. (2010) Hsp90 is required for transfer of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol. J Biol Chem 285: 31261–31267. 21. Heiligenstein S, Eisfeld K, Sendzik T, Jimenez-Becker N, Breinig F, et al. (2006) Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD. Embo J 25: 4717–4727. 22. Surjit M, Jameel S, Lal SK (2007) Cytoplasmic localization of the ORF2 protein of hepatitis E virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum. J Virol 81: 3339–3345. 23. Duriez M, Rossignol JM, Sitterlin D (2008) The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ERassociated degradation pathway. The Journal of biological chemistry 283: 32352–32360. 24. Giodini A, Cresswell P (2008) Hsp90-mediated cytosolic refolding of exogenous proteins internalized by dendritic cells. Embo J 27: 201–211. 25. Afshar N, Black BE, Paschal BM (2005) Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol. Mol Cell Biol 25: 8844–8853. 26. Petris G, Vecchi L, Bestagno M, Burrone OR (2011) Efficient Detection of Proteins Retro-Translocated from the ER to the Cytosol by In Vivo Biotinylation. PLoS One 6: e23712. 27. Li S, Spooner RA, Allen SC, Guise CP, Ladds G, et al. (2010) Foldingcompetent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum. Mol Biol Cell 21: 2543– 2554. 28. Endo Y, Mitsui K, Motizuki M, Tsurugi K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem 262: 5908–5912. 29. Redmann V, Oresic K, Tortorella LL, Cook JP, Lord M, et al. (2011) Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors. J Biol Chem 286: 21231–21238. 30. Bernardi KM, Williams JM, Kikkert M, van Voorden S, Wiertz EJ, et al. (2010) The E3 ubiquitin ligases Hrd1 and gp78 bind to and promote cholera toxin retro-translocation. Molecular biology of the cell 21: 140–151. 31. Garred O, van Deurs B, Sandvig K (1995) Furin-induced cleavage and activation of Shiga toxin. J Biol Chem 270: 10817–10821. 32. Endo Y, Tsurugi K, Yutsudo T, Takeda Y, Ogasawara T, et al. (1988) Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem 171: 45–50. 33. Johannes L, Ro¨mer W (2010) Shiga toxins–from cell biology to biomedical applications. Nat Rev Microbiol 8: 105–116. 34. Yu M, Haslam DB (2005) Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3. Infect Immun 73: 2524–2532. 35. Falguieres T, Johannes L (2006) Shiga toxin B-subunit binds to the chaperone BiP and the nucleolar protein B23. Biol Cell 98: 125–134. 36. LaPointe P, Wei X, Gariepy J (2005) 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. J Biol Chem 280: 23310–23318. 37. Saleh MT, Ferguson J, Boggs JM, Gariepy J (1996) Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes. Biochemistry 35: 9325–9334. 38. Menikh A, Saleh MT, Gariepy J, Boggs JM (1997) Orientation in lipid bilayers of a synthetic peptide representing the C-terminus of the A1 domain of shiga toxin. A polarized ATR-FTIR study. Biochemistry 36: 15865–15872. 39. Strahl-Bolsinger S, Gentzsch M, Tanner W (1999) Protein O-mannosylation. Biochim Biophys Acta 1426: 297–307. 40. Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S (2000) Structurefunction analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p. J Biol Chem 275: 19288–19296. 41. Girrbach V, Strahl S (2003) Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves. J Biol Chem 278: 12554–12562. 42. Hirayama H, Fujita M, Yoko-o T, Jigami Y (2008) O-mannosylation is required for degradation of the endoplasmic reticulum-associated degradation substrate Gas1*p via the ubiquitin/proteasome pathway in Saccharomyces cerevisiae. J Biochem 143: 555–567. 43. Gauss R, Sommer T, Jarosch E (2006) The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. Embo J 25: 1827–1835. 44. Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, et al. (2000) Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p. J Cell Biol 151: 69–82. 45. Carroll SM, Hampton RY (2010) Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase. J Biol Chem 285: 5146–5156. 46. Goder V, Carvalho P, Rapoport TA (2008) The ER-associated degradation component Der1p and its homolog Dfm1p are contained in complexes with distinct cofactors of the ATPase Cdc48p. FEBS Lett 582: 1575–1580. 47. Taxis C, Hitt R, Park SH, Deak PM, Kostova Z, et al. (2003) Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD. J Biol Chem 278: 35903–35913. 48. Kondo H, Rabouille C, Newman R, Levine TP, Pappin D, et al. (1997) p47 is a cofactor for p97-mediated membrane fusion. Nature 388: 75–78. 49. Latterich M, Frohlich KU, Schekman R (1995) Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell 82: 885–893. 50. Kim I, Ahn J, Liu C, Tanabe K, Apodaca J, et al. (2006) The Png1-Rad23 complex regulates glycoprotein turnover. J Cell Biol 172: 211–219. 51. Heinemeyer W, Kleinschmidt JA, Saidowsky J, Escher C, Wolf DH (1991) Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival. Embo J 10: 555–562. 52. Schuberth C, Buchberger A (2005) Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation. Nat Cell Biol 7: 999–1006. 53. Wilson JD, Liu Y, Bentivoglio CM, Barlowe C (2006) Sel1p/Ubx2p participates in a distinct Cdc48p-dependent endoplasmic reticulum-associated degradation pathway. Traffic 7: 1213–1223. 54. Biederer T, Volkwein C, Sommer T (1997) Role of Cue1p in ubiquitination and degradation at the ER surface. Science 278: 1806–1809. 55. Bazirgan OA, Hampton RY (2008) Cue1p is an activator of Ubc7p E2 activity in vitro and in vivo. J Biol Chem 283: 12797–12810. 56. Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Molecular biology of the cell 9: 209–222. 57. Kim W, Spear ED, Ng DT (2005) Yos9p detects and targets misfolded glycoproteins for ER-associated degradation. Mol Cell 19: 753–764. 58. Szathmary R, Bielmann R, Nita-Lazar M, Burda P, Jakob CA (2005) Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19: 765–775. 59. Quan EM, Kamiya Y, Kamiya D, Denic V, Weibezahn J, et al. (2008) Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. Mol Cell 32: 870–877. 60. Xie W, Kanehara K, Sayeed A, Ng DT (2009) Intrinsic conformational determinants signal protein misfolding to the Hrd1/Htm1 endoplasmic reticulum-associated degradation system. Mol Biol Cell 20: 3317–3329. 61. Clerc S, Hirsch C, Oggier DM, Deprez P, Jakob C, et al. (2009) Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. J Cell Biol 184: 159–172. 62. Tran JR, Tomsic LR, Brodsky JL (2011) A Cdc48p-associated factor modulates endoplasmic reticulum-associated degradation, cell stress, and ubiquitinated protein homeostasis. J Biol Chem 286: 5744–5755. 63. Rumpf S, Jentsch S (2006) Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone. Mol Cell 21: 261–269. 64. Sato BK, Schulz D, Do PH, Hampton RY (2009) Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase. Mol Cell 34: 212–222. 65. Carvalho P, Stanley AM, Rapoport TA (2010) Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143: 579–591. 66. Plemper RK, Bordallo J, Deak PM, Taxis C, Hitt R, et al. (1999) Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retrotranslocation complex mediating protein transport for ER degradation. J Cell Sci 112 (Pt 22): 4123–4134. 67. Harty C, Strahl S, Romisch K (2001) O-mannosylation protects mutant alphafactor precursor from endoplasmic reticulum-associated degradation. Mol Biol Cell 12: 1093–1101. 68. Nakatsukasa K, Okada S, Umebayashi K, Fukuda R, Nishikawa S, et al. (2004) Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast. J Biol Chem 279: 49762–49772. 69. Sandvig K, Ryd M, Garred O, Schweda E, Holm PK, et al. (1994) Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP. J Cell Biol 126: 53–64. 70. Smith DC, Sillence DJ, Falguieres T, Jarvis RM, Johannes L, et al. (2006) The association of Shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for a cytotoxic effect. Mol Biol Cell 17: 1375–1387. 71. Tam PJ, Lingwood CA (2007) Membrane cytosolic translocation of verotoxin A1 subunit in target cells. Microbiology 153: 2700–2710. 72. Day PJ, Pinheiro TJ, Roberts LM, Lord JM (2002) Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer. Biochemistry 41: 2836–2843. 73. Bar-Nun S (2005) The role of p97/Cdc48p in endoplasmic reticulum-associated degradation: from the immune system to yeast. Curr Top Microbiol Immunol 300: 95–125. 74. Aletrari MO, McKibbin C, Williams H, Pawar V, Pietroni P, et al. (2011) Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways. PLoS One 6: e22713. 75. Hassink GC, Barel MT, Van Voorden SB, Kikkert M, Wiertz EJ (2006) Ubiquitination of MHC class I heavy chains is essential for dislocation by human cytomegalovirus-encoded US2 but not US11. The Journal of biological chemistry 281: 30063–30071. 76. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/singlestranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87– 96. 77. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27. 78. Bernsel A, Viklund H, Hennerdal A, Elofsson A (2009) TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 37: W465–468. 79. Deak PM, Wolf DH (2001) Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation. J Biol Chem 276: 10663–10669. 80. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–795. 81. Vogel JP, Lee JN, Kirsch DR, Rose MD, Sztul ES (1993) Brefeldin A causes a defect in secretion in Saccharomyces cerevisiae. J Biol Chem 268: 3040–3043.
URI:	http://wrap.warwick.ac.uk/id/eprint/49232

Request changes to a record

Actions (login required)

View Item