Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif

Abstract

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein. Both are enriched in CCV coats, and further analysis of the brain-enriched isoform, NECAP 1, shows its partial localization to clathrin-coated pits and direct binding to the globular ear domain of the alpha-adaptin subunit (alpha-ear) of the adaptor protein 2 (AP-2) complex. Intriguingly, this interaction is mediated by a new motif, WVQF, that uses a distinct alpha-ear interface relative to known alpha-ear-binding partners. Disruption of this interaction blocks clathrin-mediated endocytosis. Together, our studies identify a new family of endocytic proteins that define a unique AP-2-binding motif.

Figure 1

Identification and tissue distribution of NECAPs. (A) Alignment of murine NECAP 1 and 2 with the Drosophila NECAP orthologue (dNECAP, gi 7293286). Identical and conserved amino acids are shaded black and grey, respectively. Yellow boxes denote peptides identified by tandem mass spectrometry. The sequence data are available from GenBank/EMBL/DDBJ under accession numbers and . The level of (B) NECAP 1 and (C) NECAP 2 in the indicated tissues was determined by western blot. The asterisk indicates NECAP 1 expression in brain, which is detected weakly by the NECAP 2 antibody. NECAP, adaptin-ear-binding coat-associated protein.

Figure 2

NECAP 1 in endocytic protein complexes. (A) Subcellular fractions from a preparation leading to highly enriched CCVs were western blotted with NECAP 1 and 2 and clathrin heavy-chain (CHC) antibodies. CCVs were stripped to obtain coats and vesicles. (B) NECAP 1 polyclonal antibody or pre-immune NECAP 1 sera were incubated with cytosolic or solubilized membrane fractions prepared from rat brain. Antibody was precipitated by the addition of protein-A–sepharose beads, and immunoprecipitated proteins were processed for western blotting with the antibodies indicated. Starting material (SM) is an aliquot of extract equal to one-tenth of that used for immunoprecipitation. (C) NECAP 1 immunoprecipitations were performed from Triton X-100-solubilized rat brain extract as in (B), with the addition of a protein aliquot of the unbound material (void) equal to that of the SM analysed in parallel. (D) COS-7 cells were transfected with Flag-tagged NECAP 1 and processed by indirect immunofluorescence with Flag-epitope polyclonal antibody (red) and monoclonal antibodies against clathrin (X22) and AP-2 (α-adaptin, AP.6) (green). Moderately transfected cells were analysed. Colocalization (yellow) is visualized through superimposition (merge). Higher-magnification images (merge magnified) are from the boxed areas in the merge. Scale bars, 20 μm (merge) and 5 μm (merge magnified). CCV, clathrin-coated vesicles; cpCCV, cushion pellet clathrin-coated vesicles; H, homogenate; NECAP, adaptin-ear-binding coat-associated protein; P, pellet; S, supernatant; SGp, sucrose gradient pellet; SGs, sucrose gradient supernatant.

Figure 3

NECAP 1 functions in endocytosis. (A) Domain models of NECAP 1 deletion constructs. Amino acid numbers are indicated on top. (B) Glutathione S-transferase (GST), GST–NECAP 1 and GST–NECAP 1 deletion constructs were pre-coupled to glutathione–sepharose and incubated with soluble rat brain extracts. Affinity-selected α-adaptin was detected by western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analysed in parallel. (C) COS-7 cells were transfected with Flag-tagged constructs encoding NECAP 1 and NECAP 1 deletion constructs as indicated. Endocytosis was assayed by monitoring the uptake of Cy3-labelled transferrin (Tfn). Scale bar, 20 μm. (D) Quantification of the percentage of cells that failed to take up transferrin (mean ± s.e.m., n = 4). NECAP 1, adaptin-ear-binding coat-associated protein 1.

Figure 4

Identification of a new AP-2-binding motif in NECAP 1. (A) Coat proteins stripped from clathrin-coated vesicles were separated on SDS–PAGE and transferred to nitrocellulose. Nitrocellulose strips were overlaid with glutathione S-transferase (GST) or GST–NECAP 1, or western blotted with α-adaptin and γ-adaptin antibodies. (B) GST fusion proteins encoding the ear domains of α-, β2- and γ-adaptin and GST alone were pre-coupled to glutathione–sepharose and incubated with purified His6-tagged NECAP 1. Affinity-selected His–NECAP 1 was detected with the His6 epitope tag antibody. The starting material (SM) is an aliquot of His–NECAP 1 equal to one-tenth of that added to the fusion proteins. (C) GST fusion proteins encoding full-length NECAP 1, NECAP 1 lacking the last six amino acids (Δ270–275), or the last six amino acids of NECAP 1 alone (aa 270–275), as well as GST, were pre-coupled to glutathione–sepharose and incubated with soluble rat brain extracts or purified His–α-ear. Affinity-selected proteins, along with an aliquot of the SM equal to one-tenth of that added to the fusion proteins, were western blotted with antibodies against α- and γ-adaptin or the His6 epitope tag. (D) Equimolar amounts of the indicated GST fusion proteins were pre-coupled to glutathione–sepharose and incubated with soluble rat brain extracts. Affinity-selected proteins were processed as in (C). (E) Equimolar amounts of the indicated GST fusion proteins were pre-coupled to glutathione–sepharose and incubated with 0.5 mg of soluble rat brain extract without (−) or in the presence of increasing concentrations of a peptide encoding the last 11 amino acids of NECAP 1. The molar ratio of the peptide to fusion protein is indicated. Affinity-selected proteins were western blotted as described in (C). (F) Monoclonal antibody to α-adaptin (AP.6) was incubated with a Triton X-100-solubilized rat brain extract. Antibody was precipitated by the addition of protein-G–agarose beads and immunoprecipitated proteins were processed for western blotting with the antibodies indicated. The SM is an aliquot of extract equal to one-tenth of that used for immunoprecipitation. Mock samples were treated identically, except that AP.6 antibody was excluded. (G) Model of distinct modes of interaction with α-ear. NECAP 1, adaptin-ear-binding coat-associated protein 1.