Interaction of Auxilin with the Molecular Chaperone, Hsc70

Abstract

We have studied the direct interaction of the constitutive isoform of Hsp70 (Hsc70) with the DnaJ homolog, auxilin, a cofactor that binds to clathrin-coated vesicles and is required for their uncoating by Hsc70. Auxilin caused a 5-fold increase in Hsc70 ATPase activity and a corresponding increase in steady-state levels of bound ADP; the dissociation constant for this effect was 0.6 M. Auxilin also induced polymerization of Hsc70 and bound to the resulting polymer at a 1:1 molar ratio; here too the dissociation constant was 0.6 M. Both this binding and polymerization required ATP; the Hsc70 depolymerized with a 4-min half-life when ATP was completely hydrolyzed to ADP. Although auxilin induces polymerization stoichiometrically and other DnaJ homologs induce polymerization catalytically, these data show that auxilin is similar to other DnaJ homologs in its ability to activate the Hsc70 ATPase activity, to polymerize Hsc70, and in the nucleotide dependence of this polymerization. Furthermore, the 70-amino acid J-domain of auxilin polymerized Hsc70 with the same nucleotide dependence as intact auxilin. Therefore, although only auxilin and not other DnaJ homologs support uncoating, our data suggest that various DnaJ homologs share a common mechanism of interaction with Hsc70, perhaps because their J-domains interact similarly with Hsc70.

A Functional DnaK Dimer Is Essential for the Efficient Interaction with Hsp40 Heat Shock Protein

“…Class I (DnaJ-like Hsp40s) and II have the J-domain at the N terminus and form stable dimers in solution. In contrast, class III Hsp40s do not always form dimers, such as auxilin (69,70). It is possible that the Hsp70-ATP dimer is only needed for working with class I and II Hsp40s.…”

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

“…The interaction between the J domain of auxilin and Hsc70 is required for disassembly (16), but the binding of Hsc70 to auxilin occurs at a 1∶1 ratio (Table S1) (7,10), so how does a single auxilin interact with three Hsc70 molecules? Our proposal that three Hsc70s are recruited and hydrolyze ATP in series would enable a single auxilin to stimulate the ATP hydrolysis by each Hsc70 in turn.…”

“…Grids were imaged using a Jeol 2011 transmission electron microscope with LaB6 filament. Multiple images (10)(11)(12)(13)(14)(15) were obtained for each grid, each from a different grid section, at a magnification of 10,000×. The number of cages per image were counted and averaged.…”

“…Given that auxilin and Hsc70 are known to interact at a 1∶1 stoichiometry in solution (7,10), this model raises an important question: By what mechanism can a single auxilin recruit three Hsc70 molecules?…”

“…Monitoring of cage disassembly in vitro has allowed the disassembly of clathrin cages into individual clathrin triskelions by Hsc70 and auxilin to be well-characterized in biochemical terms. Through the work of a number of different groups, the essential domains of auxilin and Hsc70 required for clathrin disassembly have been established (7,8), affinities of Hsc70 for auxilin (9,10) and nucleotides (11,12) have been determined, and the stoichiometric relationships between clathrin, Hsc70 and auxilin during disassembly have been investigated. It has been proposed that three molecules of Hsc70 are involved in removing one triskelion from a coated vesicle (13).…”