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Clathrin Functions in the Absence of the Terminal Domain Binding Site for Adaptor-associated Clathrin-Box Motifs
Authors:John R. Collette  Richard J. Chi  Douglas R. Boettner  Isabel M. Fernandez-Golbano  Rachael Plemel  Alex J. Merz  Maria Isabel Geli  Linton M. Traub  Sandra K. Lemmon
Affiliation:*Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL 33101; ;Department of Cellular Biology, Institute of Molecular Biology of Barcelona (IBMB-CSIC), 08028 Barcelona, Spain; ;§Department of Biochemistry, University of Washington, Seattle, WA 98195; and ;Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261
Abstract:Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed β-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of α-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to interact with the endocytic adaptor Ent2 in a CBM-dependent manner, and HCs encoded by chc1-box formed clathrin-coated vesicles. These data suggest that additional or alternative binding sites exist on the TD propeller to help facilitate the recruitment of clathrin to sites of vesicle formation.
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