Molecular Characterization of Endoplasmic Reticulum Deficient-2 (ERD2) in Toxoplasma gondii

Doctoral Dissertation
Thumbnail

Abstract

Toxoplasma gondii is an economically important zoonotic parasite. Prevalence of Toxoplasma has been seen worldwide. In general infection of this parasite is common in humans and may result in fatal disease, especially in immunocompromised individuals. The infectivity of this parasite mainly depends upon its coordinated protein secretion from specialized apical organelles (micronemes, dense granules and rhoptries) and maintaining the protein homeostasis is very important for parasite survival. Very few studies have been done to characterize retrograde transport between the Golgi and the ER in Toxoplasma. The study of this thesis discusses on molecular characterization of Endoplasmic reticulum deficient mutant-2 (ERD2) in Toxoplasma gondii. ERD2 receptors help in maintaining protein homeostasis by retrieving mistargeted ER luminal proteins from the post ER compartment (mainly the Golgi) to the ER. Studies were conducted to characterize classical and novel ERD2 receptors by utilizing bioinformatics analysis, drug treatments, microscopic studies, co-transfectional studies, mutational analysis, functional complementation assays and knock out strategies. For the characterization of classical ERD2, we have done pilot study where we reported the initial characterization on residues important for TgERD2 function. We observed mutation of residues predicted to be neutral in mammalian ERD2, are important in T. gondii for both location and function. In addition, we also noticed mutation of carboxyl-terminal aspartate (D) essential for retrograde transport of mammalian ERD2 cannot be successfully expressed in T. gondii phenotypes compared to homologous mutations in mammalian ERD2, suggesting a fundamental difference in folding of the protein or function of the residue. For the characterization of novel ERD2, we have shown that TgPERl-51 (TgP51) possesses the unique bipartite sequence characteristic of some apicoplast-targeted proteins and TgP51-GFP/mCherry localization overlaps with the apicoplast region. Bioinformatics analysis also reveals a novel anchor domain in TgP51, a domain that until recently has only been associated with plastid-targeted proteins in Euglena and dinoflagellates. We demonstrated that unlike TgERD2, Tg51 does not complement the yeast knockout. TgP51 localizes in BFA sensitive manner. We also show that knockout of Tgp51 gene in Ku80cells is Lethal in a “delayed death” phenotype and knock out parasites display abnormal apicoplast morphology in comparison to controls. Lastly, we propose a model for probable role of PERL in retrieval mechanism. In T. gondii, the PERLs are structurally similar to TgERD2, which functions in retrograde transport of proteins from the Golgi back to the ER. Yet, the PERLs do not appear to bind classical ERD-ligands with the same efficacy. We hypothesized that the PERLs recognize a related but slightly different motif that occurs in proteins bound for the apicoplast. Thus far our hypothesis has been supported by both a phylogenetic analysis of three chaperone protein families and the use of profile hidden Markov models. We have identified C-terminal motifs common across apicomplexa and rare among metazoa using these tools. We believe that the two positions upstream of the X may play a role in binding and may differentiate PERL and ERD binding proteins.

Attributes

Attribute NameValues
URN
  • etd-07122011-123248

Author Abhishek Tripathi
Advisor Dr. Kristin Hager
Contributor Dr. Kristin Hager, Committee Chair
Degree Level Doctoral Dissertation
Degree Discipline Biological Sciences
Degree Name PhD
Defense Date
  • 2011-06-30

Submission Date 2011-07-12
Country
  • United States of America

Subject
  • protein secretion

  • apicoplast

  • ERD2

  • retrograde

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility Public
Content License
  • All rights reserved

Departments and Units

Files

Please Note: You may encounter a delay before a download begins. Large or infrequently accessed files can take several minutes to retrieve from our archival storage system.