A Retromerlike Complex Is a Novel Rab7 Effector That Is Involved in the Transport of the Virulence Factor Cysteine Protease in the Enteric Protozoan ParasiteEntamoeba histolytica

Abstract

Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex. INTRODUCTIONRab GTPases play an essential role to regulate intracellular membrane trafficking. The compartmentalization and functions of Rab proteins are modulated at multiple layers of mechanisms including isoprenylation of the carboxy terminus, nucleotide exchange, and binding of specific effector molecules (Stenmark and Olkkonen, 2001;Takai et al., 2001;Tuvim et al., 2001). Among these modulators, effectors play key roles in the control of 7-90 Rab proteins depending on organisms from fission yeast and amoeba to mammals and plants. A variety of effectors have been identified and shown to interact with specific Rab protein. For instance, regulatory secretions of neurotransmitters from neurons or insulin from pancreatic ␤-cells are regulated by the specific interaction of Rab3 with its effectors (Jahn and Sudhof, 1999). At least four proteins, Rabphilin3, Rim1, Rim2, and Noc2, are known to bind Rab3 and recruit cAMP-GEFII, 14 -3-3, and zyxin, respectively, to regulate cAMP responsiveness, phosphoserine-dependent signaling, and the interaction with cytoskeleton (Kotake et al., 1997;Ozaki et al., 2000;Sun et al., 2003). The specificity of the Rab-effector interaction is attributable to primary sequence motifs in only a few cases including exophilins or Slip/Slac2, Rab3, and Rab27 effectors (Izumi et al., 2003). However, the majority of Rab effectors lack recognizable conserved binding motifs. For instance, Rab5 effectors, rabaptin-5, Rabex-5, EEA1, Rabenosyn-5, hVps34/p150, p110␤/p35␣, rabip4Ј, and rabankyrin-5, which a...

Evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode

“…Functional characterization in the amoebae (Bogdanovic et al, 2002;Nakada-Tsukui et al, 2005), chromalveolates such as Toxoplasma, Plasmodium and the ciliates (Ngo et al, 2003;Schilde et al, 2006;Struck et al, 2005), excavates such as kinetoplastids (Besteiro et al, 2006;Morgan et al, 2002) and Giardia (Lujan and Touz, 2003) and plants further support the idea that the LCEA had a complex membrane trafficking system. These studies have several implications for our understanding of the biology of membrane trafficking.…”

“…These include components of the three major coats (COPI, COPII and clathrin), the four SNARE families, GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), and NSF ATPases (Dacks and Doolittle, 2001;Dacks and Field, 2004;Hartman and Fedorov, 2002;Sanderfoot, 2007;Yoshizawa et al, 2006). Comparative genomics has similarly confirmed the presence, in a wide variety of eukaryotes, of a complex endocytic system, including the ESCRT complexes (Field et al, 2006), GTPases (Jekely, 2003), and homologues of the retromer complex including Vps35, Vps29 and Vps26 (Dacks et al, 2003;Damen et al, 2006;Nakada-Tsukui et al, 2005), which implies that all of these components must also have been established at a very early stage.…”

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

“…Although PX-domaincontaining proteins have been reported for P. falciparum and G. intestinalis (Banerjee et al, 2010), these do not look like sorting nexins by reverse BLAST, apart from a putative example in Giardia (GL50803_16548). Recent evidence suggests that endosome recruitment of the cargo-selective retromer subcomplex might involve factors other than the sorting nexins, notably Rab7 (Nakada- Tsukui et al, 2005;Rojas et al, 2008;Seaman et al, 2009). Rab7 is conserved in all species studied herein that lack sorting nexins, except for G. intestinalis, which might be due to either high sequence divergence or to the highly unusual nature of endocytic organelles in Giardia (Abodeely et al, 2009).…”

Rab7b controls trafficking from endosomes to the TGN

“…S5). Rab7 has been shown to interact with a retromer-like complex, and it has been recently identified as a component and regulator of the retromer and to be involved in retromer recruitment to endosomes (Nakada-Tsukui et al, 2005;Rojas et al, 2008). Its localization to late endosomes, however, has been established in several studies and Rab7 is used as a marker for this part of the endosomal pathway (Chavrier et al, 1990;Harada et al, 2005).…”