Clathrin functions in the absence of the terminal domain binding site for adaptor-associated clathrin-box motifs.
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 beta-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 alpha-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.
Collette
John R
JR
Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL 33101, USA.
Chi
Richard J
RJ
Boettner
Douglas R
DR
Fernandez-Golbano
Isabel M
IM
Plemel
Rachael
R
Merz
Alex J
AJ
Geli
Maria Isabel
MI
Traub
Linton M
LM
Lemmon
Sandra K
SK
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D001665:Binding Sites; D002687:Chitin Synthase / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism*; D033922:Clathrin Heavy Chains / Q000737:chemistry* / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013696:Temperature; D021601:trans-Golgi Network / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; C544144:CHC1 protein, S cerevisiae; D002966:Clathrin; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033922:Clathrin Heavy Chains; D049452:Green Fluorescent Proteins; D002687:Chitin Synthase; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae