Contribution of Cysteines to Clathrin Trimerization Domain Stability and Mapping of Light Chain Binding

Abstract

The three-legged or triskelion shape of clathrin is critical for the formation of polyhedral lattices around clathrincoated 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-toserine 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 ( The ability of clathrin to self-assemble is tightly linked to its molecular pinwheel shape, which facilitates the formation of clathrin-coated vesicles (CCVs) involved in carrying out receptor-mediated endocytosis at both the plasma membrane and the trans-Golgi network (TGN) (1,2). The triskelionshaped clathrin molecule comprises three identical heavy chains, each with a bound light chain subunit (2) responsible for regulating clathrin assembly (3,4). The heavy chains are joined together along a stretch near their C-terminal ends to form the trimerization domain. This domain establishes the specific 3-fold symmetry between projecting legs and also serves to gather the N-terminal domains of free clathrin molecules (triskelions) as they integrate into the growing clathrin lattice during the normal course of clathrin self-assembly (5). Recent docking studies of atomic resolution clathrin sub-domains into the 21 Å cryo-EM structure of the intact clathrin cage revealed the orientation between legs emanating from each lattice vertex was invariant (6), suggesting the trimerization domain was highly stabilized.In an effort to understand the molecular basis for trimerization domain stability, we set out to probe the role of specific cysteines (1565, 1569 and 1573) closely clustered together within this region of the clathrin molecule. To facilitate these studies, recombinant hub, which is the central region of the clathrin triskelion (3), was used instead of the full-length protein. Recombinant hubs (1074-1675) are trimeric structures with the ability to self-assemble into lattice arrays closely resembling those made by whole clathrin, but cannot form completed cages because they lack the N-terminal and distal domains required to develop curvatu...

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

“…There is an established role for CLCs in inhibiting clathrin assembly (15,16), which may be explained by the orientation of CLCs on the CHC. Through a domain in the central region, CLCs bind to the proximal leg of the CHC with their C termini oriented toward the trimerization domain (17,18). The N termini may make a U-turn halfway along the proximal leg folding back toward the trimerization domain (19) or they may extend along the proximal leg (20).…”

Novel clathrin activity: developments in health and disease

“…Superpositions show that helix 7j in CTXD is not in the same spot, but is shifted by about 10 Å away from the vertex of the trimerization domain (68). This is informative because helix 7j contains a conserved cysteine (aa1573) that we previously found to contribute to the stability of the clathrin trimer (69,72). This same cysteine was also demonstrated by multi-angle light scattering to cause bovine clathrin hub (aa1074-16750) to detrimerize and to influence the cellular distribution of mutant clathrin constructs (68).…”

Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

“…Thus, we investigated whether the human TS-clathrin is also impaired in its ability to trimerize. We expressed C-terminal fragments of human clathrin heavy chain involved in trimerization (“hub” fragments) recombinantly in E. coli [40], [41]. We then purified the recombinantly expressed fragments and crosslinked them within the range of permissive to non-permissive temperatures (modified protocol of [42]).…”

“…Recombinant expression of “clathrin-hub fragments” (C-terminus of clathrin involved in trimerization) was based on the protocol described in Ybe et al and Liu et al [40], [41]. Hub-fragments were created via PCR with the primers 5′-GCCATTGGATCCAAATTTGATGTCAATACTTC-3′ and 5′- GGCTTTGGGTACAGCATGTGAAAGCTTGCGATA-3′ and wild type or TS-clathrin human cDNA serving as a template.…”