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Fig. 1. The N termini of endosomal SNAREs interact with the ENTH domain of epsinR and Ent3p. (A) Two-hybrid interactions were detected by the ability of yeast cells (L40) to grow on selective plates. A fusion of the DNA-binding domain of LexA and the N terminus of Vti1b (amino acids 1-128, pBK111), syntaxin 7 (amino acids 1-161, pBK193) and syntaxin 8 (amino acids 1-142, pBK179) interacted with ENTH domain (amino acids 1-162) of epsinR fused to the VP16 activation domain (pBK130). No interaction was found of epsinR with the VP16 alone (pVP16-3). (B) Two-hybrid interactions were detected between the ENTH domain of Ent3p (amino acids 1-172, VP16 fusion, pKW3) and Vti1p (amino acids 1-115, LexA fusion, pBK118), Pep12p (amino acids 1-200, pBK171) and Syn8p (amino acids 1-169, pBK165) but not with Ykt6p (amino acids 1-140, pNK4), Tlg1p (amino acids 1-137, pBK172) or Vam7p (amino acids 1-255, pJZ6). ENTH domain of Ent5p (amino acids 1-172, pBK160) did not interact with any of the SNAREs. (C) In vitro binding of a bacterially expressed protein consisting of the ENTH domain of Ent3p (amino acids 1-172) fused to a C-terminal Strep-tag (pKW5) to His₆-Vti1p (amino acids 1-194, pFvM112) and His₆-Pep12p (amino acids 1-268, pFvM135), but not to His₆-Tlg1p (amino acids 1-137, pFN3) or His₆-Syn8p (amino acids 1-169, pFN6). (D) Weak in vitro binding of the ENTH domain of epsinR (amino acids 1-162) fused to a C-terminal Strep-tag (pNM3) to His₆-syntaxin 7 (amino acids 1-161, pTW1) and His₆-syntaxin 8 (amino acids 1-142, pTW2). Negative control: His₆-Vti1a (amino acids 1-187, pBK39), positive control: His₆-Vti1b (amino acids 1-207, pBK38).

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