Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.
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 self-assembly 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 high-affinity 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.
Ybe
J A
JA
The G.W.Hooper Foundation, Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0552, USA.
Greene
B
B
Liu
S H
SH
Pley
U
U
Parham
P
P
Brodsky
F M
FM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002966:Clathrin / Q000096:biosynthesis* / Q000737:chemistry*; D033922:Clathrin Heavy Chains; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride / Q000494:pharmacology*
D002966:Clathrin; D011993:Recombinant Fusion Proteins; D033922:Clathrin Heavy Chains; D012965:Sodium Chloride