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

Yuki Hanadate,Yumiko Saito-Nakano,Tomoyoshi Nozaki,Kumiko Nakada-Tsukui

doi:10.3390/ijms19123831

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.

Highlights

Membrane traffic is involved in a variety of cellular processes including transport of newly synthesized proteins, membrane compartmentalization and communication between organelles [1]
In the social amoeba Dictyostelium discoideum, intracellular osmoregulation is controlled by contractile vacuole fusion with the plasma membrane under hypotonic conditions; and this process is regulated by the serial recruitment of Rab11, Rab11 effector, Rab8 and Rab8-GTPase activating protein on the contractile vacuole membrane [11]
We previously reported that EhRab8A is localized to the endoplasmic reticulum (ER) in the steady state, which differs from the Rab8 ortholog in other organisms [17,22]

Summary

Introduction

Membrane traffic is involved in a variety of cellular processes including transport of newly synthesized proteins, membrane compartmentalization and communication between organelles [1]. Together with Rab specific binding proteins, Rab GTPases regulate various trafficking steps including vesicle budding and cargo sorting from the donor membrane [3,4], vesicle transport along the cytoskeleton [5,6] and vesicle tethering and fusion with the acceptor membrane [7]. Rab and Rab coordinately control trafficking from the trans-Golgi to the plasma membrane in mammalian cells together with their effector proteins, Rab11-family interacting proteins (FIPs) and Rabin (Rab8-guanine nucleotide exchange factor). In the social amoeba Dictyostelium discoideum, intracellular osmoregulation is controlled by contractile vacuole fusion with the plasma membrane under hypotonic conditions; and this process is regulated by the serial recruitment of Rab, Rab effector, Rab and Rab8-GTPase activating protein on the contractile vacuole membrane [11]. The Rab and Rab cascade, which is mediated by Rab effector proteins, is conserved in many tissues and organisms

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Conclusion