Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport.
ABSTRACT SNARE proteins on transport vesicles and target membranes have important roles in vesicle targeting and 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 N-terminal 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.
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ABSTRACT: Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.Journal of Biological Chemistry 03/2012; 287(21):17398-407. · 4.65 Impact Factor
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ABSTRACT: Cytokinesis is the process of partitioning the cytoplasm of a dividing cell, thereby completing mitosis. Cytokinesis in the plant cell is achieved by the formation of a new cell wall between daughter nuclei using components carried in Golgi-derived vesicles that accumulate at the midplane of the phragmoplast and fuse to form the cell plate. Proteins that play major roles in the development of the cell plate in plant cells are not well defined. Here, we report that an AP180 amino-terminal homology/epsin amino-terminal homology domain-containing protein from Arabidopsis (Arabidopsis thaliana) is involved in clathrin-coated vesicle formation from the cell plate. Arabidopsis Epsin-like Clathrin Adaptor1 (AtECA1; At2g01600) and its homologous proteins AtECA2 and AtECA4 localize to the growing cell plate in cells undergoing cytokinesis and also to the plasma membrane and endosomes in nondividing cells. AtECA1 (At2g01600) does not localize to nascent cell plates but localizes at higher levels to expanding cell plates even after the cell plate fuses with the parental plasma membrane. The temporal and spatial localization patterns of AtECA1 overlap most closely with those of the clathrin light chain. In vitro protein interaction assays revealed that AtECA1 binds to the clathrin H chain via its carboxyl-terminal domain. These results suggest that these AP180 amino-terminal homology/epsin amino-terminal homology domain-containing proteins, AtECA1, AtECA2, and AtECA4, may function as adaptors of clathrin-coated vesicles budding from the cell plate.Plant physiology 05/2012; 159(3):1013-25. · 6.56 Impact Factor
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ABSTRACT: SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on transport vesicles and target membranes are crucial for vesicle targeting and fusion. They form SNARE complexes, which contain four α-helical SNARE motifs contributed by three or four different SNAREs. Most SNAREs function only in a single transport step. The yeast SNARE Vti1p participates in four distinct SNARE complexes in transport from the trans Golgi network to late endosomes, in transport to the vacuole, in retrograde transport from endosomes to the trans Golgi network and in retrograde transport within the Golgi. So far, all vti1 mutants investigated had mutations within the SNARE motif. Little is known about the function of the N-terminal domain of Vti1p, which forms a three helix bundle called Habc domain. Here we generated a temperature-sensitive mutant of this domain to study the effects on different transport steps. The secondary structure of wild type and vti1-3 Habc domain was analyzed by circular dichroism spectroscopy. The amino acid exchanges identified in the temperature-sensitive vti1-3 mutant caused unfolding of the Habc domain. Transport pathways were investigated by immunoprecipitation of newly synthesized proteins after pulse-chase labeling and by fluorescence microscopy of a GFP-tagged protein cycling between plasma membrane, early endosomes and Golgi. In vti1-3 cells transport to the late endosome and assembly of the late endosomal SNARE complex was blocked at 37°C. Retrograde transport to the trans Golgi network was affected while fusion with the vacuole was possible but slower. Steady state levels of SNARE complexes mediating these steps were less affected than that of the late endosomal SNARE complex. As different transport steps were affected our data demonstrate the importance of a folded Vti1p Habc domain for transport.PLoS ONE 01/2013; 8(6):e66304. · 3.73 Impact Factor