Auxilin depletion causes self-assembly of clathrin into membraneless cages in vivo.
Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an approximately 50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an approximately 1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.
Hirst
Jennifer
J
Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust, Addenbrooke's Hospital, Cambridge, UK. jh228@cam.ac.uk
Sahlender
Daniela A
DA
Li
Sam
S
Lubben
Nienke B
NB
Borner
Georg H H
GH
Robinson
Margaret S
MS
D034021:Auxilins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D036681:Fluorescence Recovery After Photobleaching; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D034021:Auxilins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D034741:RNA, Small Interfering