Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges

Abstract

Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin selfassembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of highaffinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation.

Conformation of a Clathrin Triskelion in Solution

“…However, as previously mentioned, basket formation is effected by attractive interactions between clathrin legs that are resisted by leg bends and other triskelion shape changes. Thus, our failure to find a significant conformational change is important, because it suggests that lowering the pH may affect the mechanical properties of the triskelia (e.g., the rigidity) or enhance interleg interactions that favor basket formation [e.g., by changing the ionization state of histidine groups (28)]. …”

Huntingtin-interacting Protein 1 (Hip1) and Hip1-related Protein (Hip1R) Bind the Conserved Sequence of Clathrin Light Chains and Thereby Influence Clathrin Assembly in Vitro and Actin Distribution in Vivo

“…Most known regulatory and adaptor proteins influence coat assembly, membrane association, membrane fission, and uncoating by binding to sites on the HCs (1, 2), which themselves can self-assemble into a lattice. The LC subunits suppress spontaneous lattice assembly in vitro and thus allow cellular clathrin assembly to be controlled and subjected to regulation by additional proteins (3,4). Other roles for the LCs have not yet been clearly defined, despite the fact that LCs appear to be composed of multiple functional domains (5).…”

“…Near the N termini of LCs there is a 22-residue sequence, shared by LCa and LCb and completely conserved in all vertebrate LCs, which is also highly conserved in LCs from non-vertebrate species (5). The first three residues of this conserved sequence are a triplet of negatively charged residues (EED) that regulate clathrin assembly (4). If these residues are neutralized by mutation, the LCs no longer suppress in vitro clathrin assembly (4).…”

“…The first three residues of this conserved sequence are a triplet of negatively charged residues (EED) that regulate clathrin assembly (4). If these residues are neutralized by mutation, the LCs no longer suppress in vitro clathrin assembly (4). Residues to the N-terminal side of the conserved sequence in LCb are the target for casein kinase phosphorylation and residues to the C-terminal side in LCa constitute a site that can be bound by Hsc70 (5), a protein involved in CCV uncoating.…”

Huntingtin Interacting Protein 1 (HIP1) Regulates Clathrin Assembly through Direct Binding to the Regulatory Region of the Clathrin Light Chain

“…In either scenario, the N-terminal region could be oriented on the proximal leg in a manner that could inhibit the self-association of CHCs necessary for assembly. Interestingly, the N termini of the CLCs contain a domain that is conserved among all isoforms and throughout evolution (21) and this domain has been implicated in the ability of CLCs to regulate clathrin assembly (22).…”