Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog

Abstract

Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite Entamoeba histolytica. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding of EhRab8A to EhCdc50 was also confirmed by reciprocal immunoprecipitation and blue-native polyacrylamide gel electrophoresis, the latter of which revealed an 87 kDa complex. Indirect immunofluorescence imaging with and without Triton X100 showed that endogenous EhCdc50 localized on the surface in the absence of permeabilizing agent but was observed on the intracellular structures and overlapped with the ER marker Bip when Triton X100 was used. Overexpression of N-terminal HA-tagged EhCdc50 impaired its translocation to the plasma membrane and caused its accumulation in the ER. As reported previously in other organisms, overexpression and accumulation of Cdc50 in the ER likely inhibited surface transport and function of the plasma membrane lipid flippase P4-ATPase. Interestingly, HA-EhCdc50-expressing trophozoites gained resistance to miltefosine, which is consistent with the prediction that HA-EhCdc50 overexpression caused its accumulation in the ER and mislocalization of the unidentified lipid flippase. Similarly, EhRab8A gene silenced trophozoites showed increased resistance to miltefosine, supporting EhRab8A-dependent transport of EhCdc50. This study demonstrated for the first time that EhRab8A mediates the transport of EhCdc50 and lipid flippase P4-ATPase from the ER to the plasma membrane.

Molecular interplays of the Entamoeba histolytica endosomal sorting complexes required for transport during phagocytosis

“…Most of proteins were reported by Loftus et al (2005). *It is also mentioned in Hanadate et al (2016). + Proteins only reported in the AmoebaDB and Uniprot databases.…”

“…Its down-regulation by small antisense RNAmediated transcriptional gene silencing remarkably reduces adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Moreover, the surface expression of several proteins presumably involved in E. histolytica target recognition, is reduced in the EhRab8A in italics gene-silenced strain, indicating that EhRab8A regulates transport of surface receptors for the prey from the ER to the PM (Hanadate et al, 2016).…”

In Vitro and In Vivo Antiamebic Activity of Iron-Targeting Polypyridine Compounds against Enteric Protozoan Parasite Entamoeba histolytica

“…The laboratory strain HM-1:IMSS cl-6 was used to assess the antiamebic activity of the low-molecular-weight compounds with metal-binding affinity, a – p (Figure S1), 1 – 4 (Figure ), and 5 – 7 (Figure S4). According to the assay described previously with some modifications, the trophozoites were seeded and cultured into 96-well plates at 0.5 × 10 4 cells/well in 280 μL of BI-S-33 medium containing the compounds and ammonium ferric citrate at various concentrations (100% = 22.8 mg/L, 50% = 11.4 mg/L, 25% = 5.7 mg/L, or 0% = 0.0 mg/L). After 48 h of cultivation, the medium was removed and attached trophozoites were incubated with 100 μL of Opti-MEM medium containing 10% WST-1 reagent for 20 min at 37 °C.…”

Diverse Physiological Functions and Regulatory Mechanisms for Signal-Transducing Small GTPases

“…Kinesin-2 was proposed to participate in the movement of Rab5-containing endosomes and their localization at the spindle, and the endosomal transport at the onset of mitosis was suggested to be required to control the timing of nuclear envelope breakdown. Hanadate et al [ 23 ] identified and characterized the non-catalytic subunit of a lipid flippase as an interacting protein of the Rab family small GTPase Rab8A in the enteric protozoan parasite Entamoeba histolytica . This study demonstrated that Rab8A mediates the transport of the lipid flippase P4-ATPase from the ER to the plasma membrane.…”