The vesicle-mediated membrane transport is a multi-step process, consisting of vesicle formation (budding), targeting, tethering and membrane fusion (Bonifacino and Glick, 2004;Jahn and Scheller, 2006). Cargo proteins are concentrated at a specialized region on the donor membrane and packed into a nascent vesicle generated by the assembly of coat proteins such as clathrin into a cage-like lattice around the budding vesicle. Adaptor protein complexes (AP) are required to recruit cargo into coated vesicles thus playing an essential role in cargo selectivity of the transport vesicle in traffic between the trans-Golgi network (TGN) and endosomes (Owen et al., 2004). Gga proteins are monomeric clathrin adaptor proteins mediating TGN to the endosome transport (Nakayama and Wakatsuki, 2003). Apart from the AP complexes and the monomeric GGA adaptors, the list is expanding to new sets of adaptors, which are specific to only a particular type of cargo or to one family of cargo (Bonifacino and Rojas, 2006).Recently, the epsin family proteins came into view as cargospecific adaptors. The ENTH (epsin N-terminal homology) domains are phosphotidylinositol binding modules present in both mammalian epsins and in their yeast homologues Ent1p to Ent4p (Duncan and Payne, 2003;Legendre-Guillemin et al., 2004). ANTH (AP180 N-terminal homology) domains are highly related to ENTH domains (Ford et al., 2001) and present in mammalian AP180 (also known as SNAP91), CALM, HIP1, Hip1R and yeast AP180, Sla2p and Ent5p. These domains bind to different phosphoinositides. In addition to ANTH or ENTH domains, these proteins also contain binding motifs for clathrin, AP or GGA allowing them to participate in clathrin-mediated budding at the TGN, endosome or at the plasma membrane. Phylogenetic analysis of ENTH domains suggested two ENTH domain branches, mammalian epsins 1-3 and yeast Ent1p and Ent2p, which are involved in endocytosis at the plasma membrane, and enthoprotin (also known as Clint and epsinR) and yeast Ent3p functioning in transport between the TGN and endosomes (Legendre-Guillemin et al., 2004). EpsinR is localized to the TGN and in endosomal membranes (Kalthoff et al., 2002;Wasiak et al., 2002) and binds to PtdIns4P (Hirst et al., 2003;Mills et al., 2003). It also binds to clathrin, AP1 and GGA2 through its C-terminal domain Mills et al., 2003;Wasiak et al., 2002). Ent3p and Ent5p are partially redundant, bind Gga proteins and AP1 and promote formation of clathrin coats at the TGN-endosome (Costaguta et al., 2006;. ENTH domains of Ent3p and Ent5p bind PtdIns(3,5)P 2 (Eugster et al., 2004;Friant et al., 2003) and PtdIns(4,5)P 2 (Narayan and Lemmon, 2006). Ent5p associates with Vps27p and together with Ent3p is required for ubiquitin-dependent protein sorting into the interior of multivesicular bodies (MVB) (Eugster et al., 2004;Friant et al., 2003). This indicates that Ent3p and Ent5p have two different functions at the TGN-endosome and in MVB.Previously, we reported that the ENTH domains of Ent3p and epsinR specifically interact wi...

Background: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds.

The ENTH (epsin N-terminal homology) domain protein Ent3p and the ANTH [AP (adaptor protein)-180 N-terminal homology] domain protein Ent5p serve as partially redundant adaptors in vesicle budding from the TGN (trans-Golgi network) in Saccharomyces cerevisiae. They interact with phosphoinositides, clathrin, adaptor proteins and cargo such as chitin synthase Chs3p and SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). In the present study, we show that ent3Δent5Δ cells displayed defects in cell separation and bud site selection. Ent3p and Ent5p were also involved in retrograde transport from early endosomes to the TGN because GFP (green fluorescent protein)-Snc1p shifted from a plasma membrane to an intracellular localization in ent3Δent5Δ cells. The C-terminal part of Ent3p was sufficient to restore retrograde transport from early endosomes to the TGN in ent3Δent5Δ cells. In contrast, the ENTH domain and the C-terminus were required for transport from the TGN to late endosomes, demonstrating that both functions are distinct. The ENTH domain of Ent3p is known to bind the N-terminal domains of the SNAREs Vti1p, Pep12p and Syn8p, which are required for fusion with late endosomes. The interaction surface between the Ent3p-related mammalian epsinR and vti1b is known. In the present paper, we show that Vti1p bound to the homologous surface patch of Ent3p. Pep12p and Syn8p interacted with the same surface area of Ent3p. However, different amino acid residues in Ent3p were crucial for the interaction with these SNAREs in two-hybrid assays. This provides the necessary flexibility to bind three SNAREs with little sequence homology but maintains the specificity of the interaction.

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid sensing TLRs from the ER to endolysosomes, where the TLRs encounter their respective ligands and become activated. Here we show that a carboxy-terminal tyrosine-based sorting motif (Yxx Φ) in UNC93B1 differentially regulates human nucleic acid sensing TLRs in a receptor- and ligand-specific manner. Destruction of the YxxΦ motif abolished TLR7, 8 and 9 activity towards nucleic acids in human B cells and monocytes, whereas TLR8 responses towards small molecules remained intact. The YxxΦ motif in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex 1 (AP1)- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of the YxxΦ motif in UNC93B1.

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