CLATHRIN SELF-ASSEMBLY IS REGULATED BY 3 LIGHT-CHAIN RESIDUES CONTROLLING THE FORMATION OF CRITICAL SALT BRIDGES

Clathrin self-assembly into a polyhedral lattice mediates membrane pro tein sorting during endocytosis and organelle biogenesis. Lattice form ation 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 fragm ents of the clathrin heavy chain, which bind clathrin light-chain subu nits and mimic the self-assembly of intact clathrin, Reconstitution of hubs using deletion and substitution mutants of the light-chain subun its revealed that the pH dependence of clathrin self-assembly is contr olled by only three acidic residues in the clathrin light-chain subuni ts, Salt inhibition of hub assembly identified two classes of salt bri dges which are involved and deletion analysis mapped the clathrin heav y-chain regions participating in their formation, These combined obser vations indicated that the negatively charged regulatory residues, ide ntified in the light-chain subunits, inhibit the formation of high-aff inity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH, In the presence of light chains, clat hrin self-assembly depends on salt bridges that form only at low pH, b ut is exquisitely sensitive to regulation, We propose that cellular cl athrin 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.