Functional Analysis of Interaction Sites on the N‐Terminal Domain of Clathrin Heavy Chain

Abstract

In clathrin-mediated membrane traffic, clathrin does not bind directly to cargo and instead binds to adaptors that mediate this function. For endocytosis, the main adaptor is the adaptor protein (AP)-2 complex, but it is uncertain how clathrin contacts AP-2. Here we tested in human cells the importance of the three binding sites that have been identified so far on the N-terminal domain (NTD) of clathrin. We find that mutation of each of the three sites on the NTD, alone or in combination, does not block clathrin/AP-2-mediated endocytosis in the same way as deletion of the NTD. We report here the fourth and final site on the NTD that is required for clathrin/AP-2-mediated endocytic function. Each of the four interaction sites can operate alone to mediate endocytosis. The observed functional redundancy between interaction sites on the NTD explains how productivity of clathrin-coated vesicle formation is ensured.

Lessons from yeast for clathrin-mediated endocytosis

“…Another clathrin box motif binding site on the TD was predicted in this study, which could explain the limited phenotypic consequences of the TD mutation. Consistent with this, studies in mammals have now identified as many as four distinct binding sites involved in clathrin-adaptor interaction in the clathrin TD and ablation of all four sites is required to significantly affect CME 29,30 .…”

Chemical-genetic disruption of clathrin function spares adaptor complex 3–dependent endosome vesicle biogenesis

“…The dispensability of clathrin function for AP-3–dependent SLMV formation suggests that interactions between AP-3 and clathrin detected in PC12 cells and other cells types may not follow a canonical association mechanism between the ear domain of the β subunit of the adaptor and clathrin terminal domain (Willox and Royle, 2012). To test this hypothesis, we focused on fibroblasts carrying the β3A mutation pearl ( Ap3b1 pe/pe ).…”

“…AP-1 and AP-2 bind to the clathrin heavy chain via the appendages of β adaptins (Kirchhausen, 2000; Bonifacino and Traub, 2003; Bonifacino and Glick, 2004; Robinson, 2004; Hirst et al. , 2011; McMahon and Boucrot, 2011; Willox and Royle, 2012). Extensive biochemical, structural, and functional evidence indicates that clathrin association with AP-1 and AP-2 is necessary for vesicle formation by these adaptors (Kirchhausen, 2000; Bonifacino and Traub, 2003; Bonifacino and Glick, 2004; Robinson, 2004; Hirst et al.…”

“…Extensive biochemical, structural, and functional evidence indicates that clathrin association with AP-1 and AP-2 is necessary for vesicle formation by these adaptors (Kirchhausen, 2000; Bonifacino and Traub, 2003; Bonifacino and Glick, 2004; Robinson, 2004; Hirst et al. , 2011; McMahon and Boucrot, 2011; Willox and Royle, 2012). In contrast, adaptor complexes AP-4 and AP-5 do not cofractionate with clathrin-coated vesicles and may function independently of clathrin, although more research on these complexes is needed to establish certainty (Hirst et al.…”

Utilizing clathrin triskelions as carriers for spatially controlled multi-protein display

“…This includes the well-characterized clathrin-binding domain (C-box: located between blades I and II), additional sites that have been identified at distinct CTD locations (Figure 2B) [19]. To identify the most suitable CBD for dual display of proteins on clathrin triskelions, the binding affinity of three different peptides derived from accessory protein sequences shown to bind to clathrin triskelions were assessed [20–23]: (i) the LLDLD-motif, also termed C-box, derived from amphiphysin and known to bind between blades I and II of the β-propeller; (ii) the PWDLW motif (W-box) derived from SNX9 and amphiphysin that binds to the top of the β-propeller; and (iii) the LLGDL motif (R-box) found in β-arrestin that binds to the pocket present between blades IV and V (Figure 2A&B). These peptide binding motifs were linked to mCherry and GFP to obtain the mCherry-Cbox, mCherry-TriCBP, and the GFP-Wbox fusion proteins (Figure 2C).…”