Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport.
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.
Chidambaram
Subbulakshmi
S
Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Heinrich-Düker Weg 12, 37073 Göttingen, Germany.
Müllers
Nina
N
Wiederhold
Katrin
K
Haucke
Volker
V
von Mollard
Gabriele Fischer
GF
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050600:SNARE Proteins; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D021601:trans-Golgi Network / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C114154:epsin