Contribution of cysteines to clathrin trimerization domain stability and mapping of light chain binding.
The three-legged or triskelion shape of clathrin is critical for the formation of polyhedral lattices around clathrin-coated vesicles. Filamentous legs radiate from a common vertex, with amino acids 1550-1615 contributed by each leg to define the trimerization domain (Liu S-H, Wong ML, Craik CS, Brodsky FM. Cell 1995; 83: 257-267). Within this amino acid stretch there are 3 cysteines at positions 1565, 1569 and 1573 which are completely conserved in higher mammals from humans to C. elegans. The cysteine-to-serine mutation at position 1573 was observed to have the largest impact on clathrin structure and self-assembly. We have also found that Cysteine 1528 located near the boundary between the proximal region and trimerization domain mediated the formation of nonproductive clathrin aggregates when bound light chain subunits were removed. However, when light chains were added back, the ability of this cysteine to form disulfide bridges between individual clathrin molecules was blocked, suggesting bound light chain interacted with Cysteine 1528 to prevent aggregation. This new information serves to map the orientation of the light chain subunit in the vicinity of the trimerization domain and supports previous models that indicate involvement of the trimerization domain in LC binding (Chen C-Y, Reese ML, Hwang PK, Ota N, Agard D, Brodsky FM. EMBO J 2002; 21: 6072-6082; Pishvaee B, Munn A, Payne GS. EMBO J 1997; 16: 2227-2239).
Ybe
Joel A
JA
Department of Biology, Indiana University, Bloomington, IN 47405, USA. jybe@bio.indiana.edu
Ruppel
Nicholas
N
Mishra
Sanjay
S
VanHaaften
Eric
E
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002966:Clathrin / Q000737:chemistry*; D033941:Clathrin Light Chains / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D004228:Dithionitrobenzoic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation
D002966:Clathrin; D033941:Clathrin Light Chains; D004228:Dithionitrobenzoic Acid; D003545:Cysteine