When Hsc70 uncoats clathrin-coated vesicles in an auxilin-and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steadystate uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This steady-state complex forms only during uncoating in the presence of APs; in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin and continues to carry out uncoating. Whether it is complexed with ATP or ADP, the steady-state complex has very different properties from the pre-steady-state complex in that it cannot be immunoprecipitated by anticlathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.Receptor-mediated endocytosis is an essential cellular process required for the rapid import of membrane-bound receptors into cells (1-3). During receptor-mediated endocytosis, clathrin triskelions polymerize and form clathrin-coated pits on the plasma membrane; similar clathrin-coated pits form on the trans-Golgi membrane (3-5). In addition to clathrin and receptors, these coated pits contain assembly proteins that both catalyze the polymerization of clathrin triskelions and bind the receptors that are localized in the coated pit. Several clathrin assembly proteins (APs) 1 have been described (6 -10) including AP1, AP2, AP3, and AP4, which are multimeric subunit complexes, and AP180 and auxilin, which are single subunit, neuronal specific assembly proteins (11,12). In addition to assembly proteins, a number of other proteins, including synaptojanin, amphyphisin, epsin, Eps15, and the small GTPase protein, Rab5-GDI, are involved in the formation and invagination of clathrin-coated pits (13-19). Phospholipids also play an important role in clathrin coat assembly and receptor recruitment (20, 21) as does dephosphorylation of the various proteins involved in the formation of clathrin-coated pits (22,23).Following invagination of the clathrin-coated pits, the GTPbinding protein dynamin plays a key role in the pinching off of the clathrin-coated pits to form clathrin-coated vesicles (4, 5). Then, once in the cytosol, the vesicles are uncoated in an ATP-dependent process by Hsc70 and its partner protein, auxilin, which is both a clathrin assembly protein...
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.
For the most frequently used two-site model, an exact binding equation is presented in terms of the total ligand concentration. This equation has been extended to analyze the spectroscopic titration experiment where the dilution of protein solution cannot be neglected, the displacement study, and the effect of non-specific binding. Thus, with a non-linear regression program, all unknown binding parameters can be determined correctly by fitting these equations to the experimental data without any data transformation. As an example of the use of the new equations, the experimental data for receptor-insulin binding were taken from literature and reanalyzed by using a non-linear regression data analysis program.
It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP2ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD+ with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP2ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.
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