Parasitology Research

, Volume 117, Issue 5, pp 1599–1611 | Cite as

Ubc7/Ube2g2 ortholog in Entamoeba histolytica: connection with the plasma membrane and phagocytosis

  • Rinki Kumari
  • Preeti Gupta
  • Swati Tiwari
Original Paper


Endoplasmic reticulum (ER)-associated degradation (ERAD) and unfolded protein response (UPR) pathways are important for quality and quantity control of membrane and secretory proteins. We have identified orthologs of ER-associated ubiquitin conjugating enzymes (E2s) Ubc6/Ube2j2 and Ubc7/Ube2g2, ubiquitin ligases (E3) Hrd1 and GP78/AMFR, and sensor of UPR, Ire1 in E. histolytica that show conservation of important features of these proteins. Biochemical characterization of the ortholog of ERAD E2, Ubc7/Ube2g2 (termed as EhUbc7), was carried out. This E2 was transcriptionally upregulated several folds upon induction of UPR with tunicamycin. Ire1 ortholog was also upregulated upon UPR induction suggesting a linked UPR and ERAD pathway in this organism. EhUbc7 showed enzymatic activity and, similar to its orthologs in higher eukaryotes, formed polyubiquitin chains in vitro and localized to both cytoplasm and membranes. However, unlike its ortholog in higher eukaryotes, it also showed localization to the plasma membrane along with calreticulin. Inactivation of EhUbc7 significantly inhibited erythrophagocytosis, suggesting a novel function that has not been reported before for this E2. No change in growth, motility, or cell-surface expression of Gal/GalNAC lectin was observed due to inactivation of EhUbc7. The protein was present in the phagocytic cups but not in the phagosomes. A significant decrease in the number of phagocytic cups in inactive EhUbc7 expressing cells was observed, suggesting altered kinetics of phagocytosis. These findings have implications for evolutionary and mechanistic understanding of connection between phagocytosis and ER-associated proteins.


Entamoeba Endoplasmic reticulum N-glycan Parasite Phagocytosis Protozoa Quality control Ubiquitin Unfolded protein response 



Endoplasmic reticulum


Endoplasmic reticulum-associated degradation


Ubiquitin conjugating enzyme


Ubiquitin ligase




Unfolded protein response



We thank Dr. Alok Bhattacharya for the gift of amoeba culture, Dr. Sudha Bhattacharya for help with northern blot, and A. K. Sahu and Prabhat Mandal (AIRF, JNU) for confocal microscopy.

Funding information

We acknowledge funding from Department of Biotechnology in the form of “program support on molecular parasitology” and Department of Science and Technology for the PURSE grant and Defense Research and Development Organization (DRDO) grant to ST. RK was supported by fellowship from the University Grants Commission. PG was supported by the DRDO grant to ST.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

436_2018_5842_MOESM1_ESM.pdf (872 kb)
ESM 1 (PDF 872 kb)
436_2018_5842_MOESM2_ESM.avi (16.9 mb)
Online Resource 8-10 Time lapse video of control (Online Resource 8), WT (Online Resource 9) and MutUbc7 (Online Resource 10) expressing E. histolytica cells. Images were taken of live cells at 5 s intervals for 10 min. Tracks of each cell is shown in different color as captured by the Trackmate plugin of Fiji. (AVI 17282 kb)
436_2018_5842_MOESM3_ESM.avi (14 mb)
ESM 2 (AVI 14372 kb)
436_2018_5842_MOESM4_ESM.avi (17.5 mb)
ESM 3 (AVI 17944 kb)


  1. Arya S, Sharma G, Gupta P, Tiwari S (2012) In silico analysis of ubiquitin/ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species. Parasitol Res 111(1):37–51Google Scholar
  2. Banerjee S, Vishwanath P, Cui J, Kelleher DJ, Gilmore R, Robbins PW, Samuelson J (2007) The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation. Proc Natl Acad Sci U S A United States 104(28):11676–11681CrossRefGoogle Scholar
  3. Biederer T, Volkwein C, Sommer T (1996) Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. EMBO J 15(9):2069–2076PubMedPubMedCentralGoogle Scholar
  4. Burroughs AM, Jaffee M, Iyer LM, Aravind L (2008) Anatomy of the E2 ligase fold: Implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation. J Struct Biol 162(2):205–218CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chao MP, Jaiswal S, Weissman-Tsukamoto R, Alizadeh AA, Gentles AJ, Volkmer J, Weiskopf K, Willingham SB, Raveh T, Park CY, Majeti R, Weissman IL (2010) Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci Transl Med 2(63):63ra94CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M (1993) Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor. Cell 74(2):357–369CrossRefPubMedGoogle Scholar
  7. Corazzari M, Gagliardi M, Fimia GM, Piacentini M (2017) Endoplasmic reticulum stress, unfolded protein response, and cancer cell fate. Front Oncol 7:78CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cox JS, Shamu CE, Walter P (1993) Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73(6):1197–1206CrossRefPubMedGoogle Scholar
  9. Das R, Liang YH, Mariano J, Li J, Huang T, King A, Tarasov SG, Weissman AM, Ji X, Byrd RA (2013) Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine. EMBO J 32(18):2504–2516CrossRefPubMedPubMedCentralGoogle Scholar
  10. Das R, Mariano J, Tsai YC, Kalathur RC, Kostova Z, Li J, Tarasov SG, McFeeters RL, Altieri AS, Ji X, Byrd RA, Weissman AM (2009) Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78. Mol Cell 34(6):674–685CrossRefPubMedPubMedCentralGoogle Scholar
  11. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72(4):431–432CrossRefPubMedGoogle Scholar
  12. Diamond LS, Mattern CTF, Bartgis IL, Daniel WA, Keister DB (1977) Viruses of E. histolytica. VI. A study of host range. In: Sepulveda B, Diamond LS (eds) Proceedings of the international conference on Amoebiasis. Instituto Mexican de Seguro Social, Mexico CityGoogle Scholar
  13. Dumoux M, Hayward RD (2016) Membrane contact sites between pathogen-containing compartments and host organelles. Biochim Biophys Acta 1861(8 Pt B):895–899CrossRefPubMedGoogle Scholar
  14. Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2(7):379–384CrossRefPubMedGoogle Scholar
  15. Furuchi T, Hwang GW, Naganuma A (2002) Overexpression of the ubiquitin-conjugating enzyme Cdc34 confers resistance to methylmercury in Saccharomyces cerevisiae. Mol Pharmacol 61(4):738–741CrossRefPubMedGoogle Scholar
  16. Gagnon E, Duclos S, Rondeau C, Rondeau C, Chevet E, Cameron PH, Steele-Mortimer O, Paiement J, Bergeron JJ, Desjardins M (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110:119–131CrossRefPubMedGoogle Scholar
  17. Grotzke JE, Cresswell P (2015) Are ERAD components involved in cross-presentation? Mol Immunol 68(2 Pt A):112-5Google Scholar
  18. Guan KL, Dixon JE (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 192:262–267CrossRefPubMedGoogle Scholar
  19. Hamann L, Nickel R, Tannich E (1995) Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica. Proc Natl Acad Sci U S A 92:8975–8979CrossRefPubMedPubMedCentralGoogle Scholar
  20. Harbut MB, Patel BA, Yeung BK, McNamara CW, Bright AT, Ballard J, Supek F, Golde TE, Winzeler EA, Diagana TT, Greenbaum DC (2012) Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design. Proc Natl Acad Sci U S A 109:21486–21491CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hiraishi H, Mochizuki M, Takagi H (2006) Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Biosci Biotechnol Biochem 70(11):2762–2765CrossRefPubMedGoogle Scholar
  22. Hitchcock AL, Auld K, Gygi SP, Silver PA (2003) A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery. Proc Natl Acad Sci U S A 100(22):12735–12740CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lester D, Farquharson C, Russell G, Houston B (2000) Identification of a family of noncanonical ubiquitin-conjugating enzymes structurally related to yeast UBC6. Biochem Biophys Res Commun 269(2):474–480CrossRefPubMedGoogle Scholar
  24. Liu S, Chen Y, Li J, Huang T, Tarasov S, King A, Weissman AM, Byrd RA, Das R (2012) Promiscuous interactions of gp78 E3 ligase CUE domain with polyubiquitin chains. Structure 20(12):2138–2150CrossRefPubMedPubMedCentralGoogle Scholar
  25. Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, Amedeo P, Roncaglia P, Berriman M, Hirt RP, Mann BJ, Nozaki T, Suh B, Pop M, Duchene M, Ackers J, Tannich E, Leippe M, Hofer M, Bruchhaus I, Willhoeft U, Bhattacharya A, Chillingworth T, Churcher C, Hance Z, Harris B, Harris D, Jagels K, Moule S, Mungall K, Ormond D, Squares R, Whitehead S, Quail MA, Rabbinowitsch E, Norbertczak H, Price C, Wang Z, Guillén N, Gilchrist C, Stroup SE, Bhattacharya S, Lohia A, Foster PG, Sicheritz-Ponten T, Weber C, Singh U, Mukherjee C, El-Sayed NM, Petri WA, Clark CG, Embley TM, Barrell B, Fraser CM, Hall N (2005) The genome of the protist parasite Entamoeba histolytica. Nature 433(7028):865–868CrossRefPubMedGoogle Scholar
  26. Marie C, Petri WA (2014) Regulation of virulence of Entamoeba histolytica. Annu Rev Microbiol 68:493–520CrossRefPubMedGoogle Scholar
  27. McCaffrey K, Braakman I (2016) Protein quality control at the endoplasmic reticulum. Essays Biochem 60(2):227–235CrossRefPubMedGoogle Scholar
  28. Mortimer L, Chadee K (2010) The immunopathogenesis of Entamoeba histolytica. Exp Parasitol 126(3):366–380CrossRefPubMedGoogle Scholar
  29. Nickel R, Tannich E (1994) Transfection and transient expression of chloramphenicol acetyltransferase gene in the protozoan parasite Entamoeba histolytica. Proc Natl Acad Sci U S A 91(15):7095–7098CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nunes-Hasler P, Demaurex N (2017) The ER phagosome connection in the era of membrane contact sites. Biochim Biophys Acta 1864(9):1513–1524CrossRefPubMedGoogle Scholar
  31. Ogden CA, deCathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, Henson PM (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 194:781–795CrossRefPubMedPubMedCentralGoogle Scholar
  32. Printsev I, Curiel D, Carraway KL 3rd (2016) Membrane protein quantity control at the endoplasmic reticulum. J Membr Biol 250:379–392CrossRefPubMedGoogle Scholar
  33. Ravid T, Hochstrasser M (2007) Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue. Nat Cell Biol 9(4):422–427CrossRefPubMedGoogle Scholar
  34. Roy CR (2002) Exploitation of the endoplasmic reticulum by bacterial pathogens. Trends Microbiol 10(9):418–424CrossRefPubMedGoogle Scholar
  35. Saito-Nakano Y, Loftus BJ, Hall N, Nozaki T (2005) The diversity of Rab GTPases in Entamoeba histolytica. Exp Parasitol 110:244–252CrossRefPubMedGoogle Scholar
  36. Samuelson J, Robbins PW (2015) Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation. Semin Cell Dev Biol 41:121–128CrossRefPubMedGoogle Scholar
  37. 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(2):212–222CrossRefPubMedPubMedCentralGoogle Scholar
  38. Scheffner M, Huibregtse JM, Howley PM (1994) Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc Natl Acad Sci U S A 91(19):8797–8801CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefPubMedGoogle Scholar
  40. Schoebel S, Mi W, Stein A, Ovchinnikov S, Pavlovicz R, DiMaio F, Baker D, Chambers MG, Su H, Li D, Rapoport TA, Liao M (2017) Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3. Nature 548(7667):352–355CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sehgal D, Mittal V, Ramachandran S, Dhar SK, Bhattacharya A, Bhattacharya S (1994) Nucleotide sequence organisation and analysis of the nuclear ribosomal DNA circle of the protozoan parasite Entamoeba histolytica. Mol Biochem Parasitol 67(2):205–214CrossRefPubMedGoogle Scholar
  42. Sommer T, Jentsch S (1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365(6442):176–179CrossRefPubMedGoogle Scholar
  43. Teixeira JE, Heron BT, Huston CD (2008) C1q- and collectin-dependent phagocytosis of apoptotic host cells by the intestinal protozoan Entamoeba histolytica. J Infect Dis 198:1062–1070CrossRefPubMedPubMedCentralGoogle Scholar
  44. Tinevez JY, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, Bednarek SY, Shorte SL, Eliceiri KW (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90CrossRefPubMedGoogle Scholar
  45. Tiwari S, Weissman AM (2001) Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitin-conjugating enzymes (E2s). J Biol Chem 276(19):16193–16200CrossRefPubMedGoogle Scholar
  46. Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, Chow A, Jiang A, Shaw J, Yip C, Moore HP, van der Wel N, Houben D, Peters PJ, de Chastellier C, Mellman I, Grinstein S (2005) Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell:123157–123170Google Scholar
  47. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101(3):249–258CrossRefPubMedGoogle Scholar
  48. Ushioda R, Hoseki J, Nagata K (2013) Glycosylation-independent ERAD pathway serves as a backup system under ER stress. Mol Biol Cell 24(20):3155–3163CrossRefPubMedPubMedCentralGoogle Scholar
  49. Vaithilingam A, Teixeira JE, Miller PJ, Heron BT, Huston CD (2012) Entamoeba histolytica cell surface calreticulin binds human c1q and functions in amebic phagocytosis of host cells. Infect Immun 80(6):2008–2018CrossRefPubMedPubMedCentralGoogle Scholar
  50. Weber A, Cohen I, Popp O, Dittmar G, Reiss Y, Sommer T, Ravid T, Jarosch E (2016) Sequential poly-ubiquitylation by specialized conjugating enzymes expands the versatility of a quality control ubiquitin ligase. Mol Cell 63(5):827–839CrossRefPubMedGoogle Scholar
  51. Wojcikiewicz RJ, Pearce MM, Sliter DA, Wang Y (2009) When worlds collide: IP(3) receptors and the ERAD pathway. Cell Calcium 46:147–153CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wu CJ, Conze DB, Li X, Ying SX, Hanover JA, Ashwell JD (2005) TNF-alpha induced c-IAP1/TRAF2 complex translocation to a Ubc6-containing compartment and TRAF2 ubiquitination. EMBO J 24(10):1886–1898CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zemoura K, Schenkel M, Acuña MA, Yévenes GE, Zeilhofer HU, Benke D (2013) Endoplasmic reticulum-associated degradation controls cell surface expression of γ-aminobutyric acid, type B receptors. J Biol Chem 288:34897–34905CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of BiotechnologyJawaharlal Nehru UniversityNew DelhiIndia
  2. 2.Microbiology DivisionDefence Research and Development EstablishmentGwaliorIndia

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