Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites.

摘要: Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds heterotetrameric AP-2 adaptor complex by associating directly with globular appendage α subunit. We have found this portion 1 also associates clathrin. interaction clathrin direct and not mediated epsin-bound AP-2. Alanine scanning mutagenesis shows binding depends on sequence 257LMDLADV located within domain. This sequence, related known clathrin-binding sequences β subunits, amphiphysin, β-arrestin, facilitates association terminal domain heavy chain. Unexpectedly, inhibiting GST-epsin fusion progressively deleting triplets but leaving LMDLADV intact, diminishes parallel Because subunit contains site, optimal soluble depend presence at least two distinct sites, we show second 480LVDLD, carboxyl-terminal segment interacts directly. LVDLD act cooperatively recruitment assays, suggesting they bind different sites clathrin-terminal evolutionary conservation similar several metazoan epsin-like molecules suggests ability establish multiple protein-protein contacts developing clathrin-coated bud aspect function.