Clathrin-coated vesicles mediate diverse processes such as nutrient uptake,
downregulation of hormone receptors, formation of synaptic vesicles, virus
entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat
assembly and disassembly are integral features of clathrin-mediated vesicu
lar transport (Fig. la). Coat assembly involves recruitment of clathrin tri
skelia, adaptor complexes and other factors that influence coat assembly, c
argo sequestration, membrane invagination and scission(1-3) (Fig. 1a). Coat
disassembly is thought to be essential for fusion of vesicles with target
membranes and for recycling components of clathrin coats to the cytoplasm f
or further rounds of vesicle formation. In vitro, cytosolic heat-shock prot
ein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassem
bly(4). However, a specific function of these factors in uncoating in vivo
has not been demonstrated, leaving the physiological mechanism and signific
ance of uncoating unclear. Here we report the identification and characteri
zation of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation o
f Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo
delivery, and an increased ratio of vesicle-associated to cytoplasmic clath
rin. Our results demonstrate an in vivo uncoating function of a J domain cc
-chaperone and establish the physiological significance of uncoating in tra
nsport mediated by clathrin-coated vesicles.