Biochemie III, Fakultät für Chemie, Universitätstrasse 25, Universität Bielefeld, 33615 Bielefeld, Germany.
Biochem J. 2010 Oct 1;431(1):123-34. doi: 10.1042/BJ20100693.
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.
ENTH（epsilonin N 端同源）结构域蛋白 Ent3p 和 ANTH（AP（衔接蛋白）-180 N 端同源）结构域蛋白 Ent5p 作为部分冗余衔接物，参与了酿酒酵母从 TGN（高尔基体网络）出芽形成囊泡的过程。它们与磷酸肌醇、网格蛋白、衔接蛋白和货物（如几丁质合成酶 Chs3p 和 SNAREs（可溶性 N-乙基马来酰亚胺敏感融合蛋白附着蛋白受体））相互作用。在本研究中，我们发现 ent3Δent5Δ 细胞在细胞分离和芽点选择中存在缺陷。Ent3p 和 Ent5p 还参与了从早期内体到 TGN 的逆行运输，因为 GFP（绿色荧光蛋白）-Snc1p 从质膜转移到 ent3Δent5Δ 细胞的细胞内定位。Ent3p 的 C 端部分足以恢复 ent3Δent5Δ 细胞中从早期内体到 TGN 的逆行运输。相比之下，ENTH 结构域和 C 末端对于从 TGN 到晚期内体的运输是必需的，这表明这两种功能是不同的。Ent3p 的 ENTH 结构域已知与 SNAREs Vti1p、Pep12p 和 Syn8p 的 N 端结构域结合，这些 SNAREs对于与晚期内体融合是必需的。Ent3p 相关的哺乳动物 epsinR 和 vti1b 之间的相互作用表面是已知的。在本文中，我们表明 Vti1p 与 Ent3p 的同源表面斑块结合。Pep12p 和 Syn8p 与 Ent3p 的同一表面区域相互作用。然而，在双杂交测定中，Ent3p 中的不同氨基酸残基对于与这些 SNAREs 的相互作用是至关重要的。这提供了与具有很少序列同源性的三个 SNAREs 结合的必要灵活性，同时保持了相互作用的特异性。
