Stonin 2 binds directly to AP2 via a novel type of α -ear binding motif

AP2 subunits from RBE efficiently. However, whether stonin 2-AP2 interaction was direct or indirectly mediated by other proteins such as eps15 remained unclear.

Eps15 contains 15 DPF motifs in its carboxy-terminal part and therefore it may link stonin 2 to the AP2 complex. In order to investigate this possibility, we first made use of the fact that eps15 can be quantitatively purified by immobilized GST-Stonin 2NT fusion protein and thereby became depleted from the extract. Surprisingly, we could detect similar amounts of AP2 in the eluate if affinity purification was done from eps15 depleted rat brain extracts (Figure 3.7). Although, the presence of unidentified bridging proteins could not be excluded, these data prompted us to investigate whether stonin 2 is able to directly bind to AP2.

Figure 3.7 Binding of stonin 2 to AP2 in eps15 depleted RBE. RBE was incubated with GST or GST-Stonin 2NT fusion protein bound agarose beads. After the incubation beads were recovered (B1) and extract (U1) was re-incubated with a fresh batch of protein bound beads (B2). B: Bound, U:

Unbound.

Many endocytic accessory proteins harbor structurally unfolded linear peptide sequences that are recognized by AP2 subunits or clathrin. DPF motifs within eps15, DPW within epsin 1 and FXDXF type motifs within amphiphysins provide examples for peptide sequences capable of association with the AP2-α-appendage domain.

The strong interaction between stonin 2 and AP2 suggested the presence of one or likely multiple AP2 interaction motifs within stonin 2. However, analysis of the stonin 2 amino acid sequence did not reveal the presence of any canonical FXDXF or DXF/W boxes .

In order to test if stonin 1 is also able to interact with AP2, we generated GST fusion constructs of full length mouse stonin 1 and its various truncations (serine/proline rich domain: 1-200aa, stonin HD+µHD: 201-730aa and µHD alone: 403-730aa). Affinity purification experiments from RBE showed that stonin 1 is capable of binding to AP2 via a putative interaction motif present within its amino-terminal first 200 amino acids encompassing the serine/proline rich domain (Figure 3.8). In contrast to stonin 2, we could not detect any significant interaction between stonin 1 and EH-domain containing proteins such as Eps15 or intersectin 1. This is likely due to the fact that stonin 1 contains only a single NPF motif insufficient for high-affinity EH-domain binding.

Figure 3.8 Interaction of stonin 1 with AP2, eps15 and intersectin 1. GST or GST-Stonin1 fusion protein bound beads were incubated with 1mg RBE by gentle mixing on a rotating wheel in order to affinity purify those proteins from RBE that interact with stonin 1. After three washes, bound proteins were eluted from the beads by boiling in sample buffer. After separation by SDS-PAGE, proteins were blotted to nitrocellulose membrane and decorated with antibodies against indicated proteins.

The amino-terminal serine/proline rich domains of stonin 1 and stonin 2 display little sequence conservation using different peptide sequence alignment algorithms. To reveal putative AP2 interaction motifs, we performed a dot plot alignment of the amino-terminal domains of stonins 1 and 2, searching for short (4-6 amino acids), nearly exact matches (>75% identity). Using these parameters we discovered a putative motif with the consensus sequence WVXFaaa with any of the amino acids being an acidic residue. (Figure 3.9). This motif is present once in stonin 1 and three times in stonin 2. Database searches for proteins harboring similar peptide sequences yielded several other endocytic accessory proteins including synaptojanin, pacsin/syndapin, and intersectin 2 . In some of these cases, AP2 interaction was known but no interaction motif had been identified (Conner et al., 2002; Haffner et al., 2000).

Figure 3.9 Novel AP2 interaction motif initially discovered in stonins. The novel AP2 interaction motif consists of three sequence components that are essential for binding to AP2; (i) a tryptophan at the first position (ii) a phenylalanine at the 4^th position (iii) one or more negatively charged residues at 5^th, 6^th and 7^th positions. Although the mutation of valine at the second position to alanine significantly inhibits AP2 binding, it is not essential (Walther et al., 2004).

In order to test for the requirement of the WVXF type motif in binding of stonin 1 to AP2 complex, we generated stonin 1 constructs with truncations lacking the single WVXF motif (residues 16-200) or containing the amino-terminal 33 residues including the WVXF sequence. A GST-Stonin 1(1-33) fusion protein but not GST-Stonin 1(16-200)

was able to affinity purify AP2 from RBE (Figure 3.10). No interaction with AP1 could be detected by Western blot analysis. Using GST-Stonin 1(1-33), we could purify chemical amounts of AP2 from RBE, that can be seen by Coomassie blue staining of SDS gels (Figure 3.10B).

Figure 3.10 Delineation of WvXF type motifs in stonin1 sequence. (A) Mouse stonin1 contains the WVTFDDD peptide sequence between residues 9-15. GST fusion proteins that include this region can affinity purify AP2 from the RBE. GST-Stonin1_(16-200) has lost the binding capability to AP2. (B) Quantitative levels of AP2 can be affinity purified from the RBE using GST-Stonin1_(1-33) construct. AP2 subunits α, β and µ2 are indicated with arrows on the figure.

Concurrent with our findings, other groups have identified the same AP2 interaction motif in NECAPs and the long-splice isoform of synaptojanin1 (Ritter et al., 2003;

Jha et al., 2003). Interestingly, NECAPs also harbor a WGDF motif just 20 amino acids upstream of the AP2 binding WVQF motif, which has been shown to bind to the TGN localized clathrin adaptor AP1 (Ritter et al., 2004). The position of the negatively charged residues determines the binding specificity of these two motifs to AP1 or AP2 such that AP1 binding requires presence of negative charges upstream of the WXXF motif and AP2 binding in the downstream (Ritter et al., 2004).

3.4. Overexpression of stonin 2 but not stonin 1 blocks CME in COS7 cells