Analysis of clathrin light chain-heavy chain interactions using truncated mutants of rat liver light chain LCB3.
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.
Scarmato
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P
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
Kirchhausen
T
T
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002872:Chromosome Deletion; D002966:Clathrin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011485:Protein Binding; D051381:Rats
D002966:Clathrin; D004274:DNA, Recombinant; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D004247:DNA