Abstract
Clathrin light chains are extended molecules located along the proximal segment of each of the three heavy chain legs of a clathrin trimer. All mammalian light chains share a central segment with 10 repeated heptad motifs believed to mediate the interaction with clathrin heavy chains. In order to test this model in more detail, we have expressed intact rat liver clathrin light chain LCB3 in Escherichia coli and find that it binds tightly to calf clathrin heavy chains. Using a set of expressed truncated mutants of LCB3, we show that the presence of seven to eight heptads is indeed necessary for a successful interaction. More extensive deletions of the central segment completely abolish the ability to bind to heavy chains. Neither the amino- nor the carboxyl-terminal domain is essential for binding, but competition experiments show that the presence of the carboxyl-terminal domain does enhance the interaction with heavy chains.
Highlights
Clathrin-coated pits and coated vesicles are organelles found in association with the plasma membrane and Golgi complex of eukaryotic cells that mediate the selective entrapment of macromolecules destined for vesicular traffic
Expression of Full-length Rat Liver Clathrin Light Chain LCB3 in E. coli-The plasmid rLCB3 directs the synthesis of two principal protein products of similar size that accumulate in the periplasm of E. coli (Fig. 1, lane 3)
It is known that light chains isolated from mammalian clathrin retain their ability to bind back to clathrin heavy chain even after exposure to temperatures higher than 95 “C [21]
Summary
Clathrin-coated pits and coated vesicles are organelles found in association with the plasma membrane and Golgi complex of eukaryotic cells that mediate the selective entrapment of macromolecules destined for vesicular traffic (reviewed in Refs. l-4). We have proposed a model in which this segment mediates binding to clathrin heavy chains while the surrounding regions mediate interactions with other proteins This model is supported by the observation that several monoclonal antibodies directed against epitopes located within the heptad region inhibit the light chainheavy chain interaction [17]. Unlike the epitopes located in the central segment, the antibodies directed against epitopes in the carboxyl-terminal segment did not block light chain-heavy chain binding. These antibodies react with native light chain-containing clathrin trimers indicating that the epitopes are accessible because they are oriented toward the aqueous environment. We find that removal of the carboxyl-terminal segment of LCB3 decreases the strength of the light chain-heavy chain interaction suggesting that this region may be required for initial anchorage of light chains to heavy chains followed by the more extensive interaction mediated by assembly of the helical segment
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