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

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Abstract

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.

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Abbreviations

ER:

Endoplasmic reticulum

ERAD:

Endoplasmic reticulum-associated degradation

E2:

Ubiquitin conjugating enzyme

E3:

Ubiquitin ligase

Ub:

Ubiquitin

UPR:

Unfolded protein response

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Acknowledgements

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

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.

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  1. Preeti Gupta

    Present address: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, MP, 474002, India

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  1. School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India

    Rinki Kumari & Swati Tiwari

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  1. Rinki Kumari
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  2. Preeti Gupta
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Correspondence to Swati Tiwari.

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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)

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Kumari, R., Gupta, P. & Tiwari, S. Ubc7/Ube2g2 ortholog in Entamoeba histolytica: connection with the plasma membrane and phagocytosis. Parasitol Res 117, 1599–1611 (2018). https://doi.org/10.1007/s00436-018-5842-6

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