Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70.
During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.
Ma
Yuchen
Y
Laboratory of Cell Biology, NHLBI/National Institutes of Health, 50 South Drive, Bethesda, MD 20892-0301, USA.
Greener
Tsvika
T
Pacold
Michael E
ME
Kaushal
Shivani
S
Greene
Lois E
LE
Eisenberg
Evan
E
D000818:Animals; D034021:Auxilins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D034021:Auxilins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins