Dissecting Ent3p: The ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemical Journal Année : 2010

Dissecting Ent3p: The ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes

Résumé

The epsin N-terminal homology (ENTH) domain protein Ent3p and the AP-180 N-terminal homology (ANTH) domain protein Ent5p serve as partially redundant adaptors in vesicle budding from the TGN in Saccharomyces cerevisiae. They interact with phosphoinositides, clathrin, adaptor proteins and cargo such as chitin synthase Chs3p and SNAREs. Here we show that ent3∆ent5∆ 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-Snc1p shifted from a plasma membrane to an intracellular localization in ent3∆ent5∆ cells. The C-terminal part of Ent3p was sufficient to restore retrograde transport from early endosomes to the TGN in ent3∆ent5∆ cells. By contrast, 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. Here 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 necessary flexibility to bind three SNAREs with little sequence homology but maintains specificity of the interaction.

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Dates et versions

hal-00517251 , version 1 (14-09-2010)

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Jana Zimmermann, Subbulakshmi Chidambaram, Gabriele Fischer von Mollard. Dissecting Ent3p: The ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes. Biochemical Journal, 2010, 431 (1), pp.123-134. ⟨10.1042/BJ20100693⟩. ⟨hal-00517251⟩

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