Gao B, Eisenberg E, Greene L
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Jul 12;271(28):16792-7. doi: 10.1074/jbc.271.28.16792.
Constitutive 70-kDa heat shock protein (hsc70) is a mixture of monomers and oligomers in ADP, while in ATP it is monomeric unless certain DnaJ homologs are present which induce hsc70 to form large polymers in an ATP-dependent reaction. A key question regarding polymerized hsc70 is whether it is able to bind protein substrates. Polymerized BiP, the hsc70 present in the endoplasmic reticulum, has been found to bind substrates in vitro although substrates appear to bind only to monomeric BiP in vivo. In this study, we investigated whether substrate binds to polymerized cytoplasmic hsc70 in vitro. Although both stoichiometric ATP and high concentrations of cytochrome c peptide monomerized hsc70, direct binding studies provided no evidence that cytochrome c peptide binds to polymerized hsc70. Furthermore, the time course of cytochrome c peptide and clathrin binding to hsc70 suggested that rather than binding to polymerized hsc70, they monomerized it by reducing free monomer, thereby shifting the monomer-polymer equilibrium toward monomer. We conclude that peptide and protein substrates bind at least an order of magnitude more weakly to polymerized hsc70 than to monomer, suggesting that polymerization of hsc70 in vivo, perhaps by DnaJ homologs, may store it in an inactive form.
组成型70 kDa热休克蛋白（hsc70）在ADP状态下是单体和寡聚体的混合物，而在ATP状态下它是单体形式，除非存在某些DnaJ同源物，这些同源物会在ATP依赖性反应中诱导hsc70形成大聚合物。关于聚合态hsc70的一个关键问题是它是否能够结合蛋白质底物。内质网中的hsc70即聚合态结合蛋白（BiP），已发现在体外它能结合底物，尽管在体内底物似乎仅与单体形式的BiP结合。在本研究中，我们调查了底物在体外是否能与聚合态的细胞质hsc70结合。尽管化学计量的ATP和高浓度的细胞色素c肽都能使hsc70单体化，但直接结合研究没有提供证据表明细胞色素c肽能与聚合态hsc70结合。此外，细胞色素c肽和网格蛋白与hsc70结合的时间进程表明，它们并非与聚合态hsc70结合，而是通过减少游离单体使hsc70单体化，从而使单体 - 聚合物平衡向单体方向移动。我们得出结论，肽和蛋白质底物与聚合态hsc70的结合力至少比与单体的结合力弱一个数量级，这表明体内hsc70的聚合（可能由DnaJ同源物介导）可能将其以无活性形式储存。
