EpsinR: an ENTH Domain-containing Protein that Interacts with AP-1

Abstract

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the ␥-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH 2 -terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTP␥S, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.

EpsinR Is an Adaptor for the SNARE Protein Vti1b

“…For pull-down experiments, GST fusion proteins were made with the NH 2 -terminal domains of epsinR (amino acids 1-166) and vti1b (amino acids 1-206) and pull-downs were carried out essentially as previously described (Hirst et al, 2003), using HeLa extracts in PBS containing 0.1% NP-40 and the protease inhibitor AEBSF, at a protein concentration of 1.5 mg/ml. Bound proteins were eluted with sample buffer and subjected to SDS-PAGE.…”

“…Rabbit antibodies against epsinR, AP-1 ␥, clathrin, CI-MPR, and 1A were raised in house and have already been described (Page et al, 1999;Hirst et al, 2003). Monoclonal antibodies were purchased from BD Transduction Laboratories (Lexington,KY;, from Santa Cruz Biotechnology (Santa Cruz, CA; anti-EF-2), from Sigma (St. Louis, MO; antig), and from Qbiogene (Carlsbad, CA; anti-CD8).…”

“…Monoclonal antibodies were purchased from BD Transduction Laboratories (Lexington,KY;, from Santa Cruz Biotechnology (Santa Cruz, CA; anti-EF-2), from Sigma (St. Louis, MO; antig), and from Qbiogene (Carlsbad, CA; anti-CD8). Western blots were probed with these antibodies, followed by a rabbit anti-mouse linker where appropriate and then by 125 I-protein A as previously described (Hirst et al, 2003). Samples included GST pull-downs, whole cell extracts, and CCVs isolated from tissue culture cells.…”

“…An epsinR sequence, AAC-CAUUGAUCUUGGAGCAGC, which had previously been found to be ineffective for knockdown experiments (Hirst et al, 2003), was used as a control.…”

“…One of these proteins, which has been variously named epsinR (Hirst et al, 2003;Mills et al, 2003), enthoprotin (Wasiak et al, 2002), and Clint (Kalthoff et al, 2002), is a particularly promising candidate, because it binds not only to AP-1, but also to clathrin and to phosphoinositides. The phosphoinositide interaction is mediated by the ENTH (epsin N-terminal homology) domain of epsinR, but unlike the ENTH domains of epsins 1-3, which bind PIP2, the ENTH domain of epsinR prefers PI(4)P, a phosphoinositide mainly generated on TGN membranes.…”