SNARE proteins on transport vesicles and target membranes have important roles in vesicle targetingand fusion. Therefore, localization and activity of SNAREs have to be tightly controlled. Regulatory proteins bind to N-terminal domains of some SNAREs. vti1b is a mammalian SNARE that functions in late endosomal fusion. To investigate the role of the N terminus of vti1b we performed a yeast two-hybrid screen. The N terminus of vti1b interacted specifically with the epsin Nterminal homology (ENTH) domain of enthoprotin/ CLINT/epsinR. The interaction was confirmed using in vitro binding assays. This complex formation between a SNARE and an ENTH domain was conserved between mammals and yeast. Yeast Vti1p interacted with the ENTH domain of Ent3p. ENTH proteins are involved in the formation of clathrin-coated vesicles. Both epsinR and Ent3p bind adaptor proteins at the trans-Golgi network. Vti1p is required for multiple transport steps in the endosomal system. Genetic interactions between VTI1 and ENT3 were investigated. Synthetic defects suggested that Vti1p and Ent3p cooperate in transport from the trans-Golgi network to the prevacuolar endosome. Our experiments identified the first cytoplasmic protein binding to specific ENTH domains. These results point toward a novel function of the ENTH domain and a connection between proteins that function either in vesicle formation or in vesicle fusion. SNARE 1 proteins on transport vesicles and target membranes mediate recognition and fusion between membranes by complex formation with other SNAREs via SNARE motifs (1). SNAREs can be subdivided into four different groups (R-, Qa-, Qb-, and Qc-SNAREs, with R and Q being the single-letter abbreviations of arginine and glutamine, respectively) according to similarities in their amino acid sequences. All well characterized SNARE complexes are composed of four different SNARE helices, one from each group. The conserved SNARE motif is located next to a C-terminal transmembrane anchor in most SNAREs. Many SNAREs contain N-terminal domains that fold independently and are highly divergent in their amino acid sequences. The N-terminal domains of SNAREs belonging to syntaxins or to Qa-SNAREs can bind proteins that regulate SNARE complex formation. Other binding partners recruit Qaor R-SNAREs into budding vesicles. The N-terminal domains of the R-SNAREs Sec22p (2) and Ykt6p (3) form a mixed ␣-helical/ ␤-sheet profilin-like fold. By contrast, the N-terminal domains of Qa-SNAREs consist of a three-helix bundle.We set out to identify interaction partners for Qb-SNAREs, which function in endosomal traffic. The yeast Qb-SNARE Vti1p is required for the following: (i) transport from the TGN to the prevacuolar endosome; (ii) traffic to the vacuole (equivalent to the mammalian lysosome); (iii) retrograde traffic to the cis-Golgi; and (iv) homotypic TGN fusion as part of four different SNARE complexes (4 -6). Mammalian cells contain two homologs of yeast Vti1p. vti1a and vti1b share 30% of their amino acid residues with each other as well as with V...

SNAREs on transport vesicles and target membranes are required for vesicle targeting and fusion. Here we describe a novel yeast protein with a typical SNARE motif but with low overall amino acid homologies to other SNAREs. The protein localized to the endoplasmic reticulum (ER) and was therefore named Use1p (unconventional SNARE in the ER). A temperature‐sensitive use1 mutant was generated. use1 mutant cells accumulated the ER forms of carboxypeptidase Y and invertase. More specific assays revealed that use1 mutant cells were defective in retrograde traffic to the ER. This was supported by strong genetic interactions between USE1 and the genes encoding SNAREs in retrograde traffic to the ER. Antibodies directed against Use1p co‐immunoprecipitated the SNAREs Ufe1p, myc‐Sec20p and Sec22p, which form a SNARE complex required for retrograde traffic from the Golgi to the ER, but neither Bos1p nor Bet1p (members of the SNARE complex in anterograde traffic to the Golgi). Therefore, we conclude that Use1p is a novel SNARE protein that functions in retrograde traffic from the Golgi to the ER.

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...

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.

PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.