Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells
Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells
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DOI：
10.1091/mbc.8.8.1559
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发表时间：
1997-08-01
影响因子：
3.3
通讯作者：
Hansen, WJ
中科院分区：
文献类型：
--
作者：
Eggers, DK;Welch, WJ;Hansen, WJ
新合成的蛋白质在体内的折叠被认为是通过一组被称为分子伴侣的蛋白质的合作相互作用来促进的。我们用多种方法研究了哺乳动物细胞胞浆中的伴侣与新生多肽的直接相互作用。一种使用嘌呤霉素多克隆抗体的新方法使我们能够标记和捕获一组截短的新生多肽，而不会对结合的伴侣蛋白的身份产生偏见。此外，还比较了识别胞浆伴侣蛋白HSP70、CCT(TRIC)、HSP40、P48(HIP)和HSP90的抗体在化学交联前后共沉淀新生多肽的能力。通过这三种方法，HSP70被发现是结合新生多肽的主要伴侣。在生理条件下，HSP70与新生多肽之间的相互作用明显是动态的，但可以通过耗尽ATP或通过交联来稳定。胞浆伴侣蛋白CCT主要与全长、新合成的肌动蛋白和微管蛋白结合。我们证明并警告，新生多肽具有与细胞裂解物中的许多蛋白质非特异性结合的倾向。尽管目前的体内蛋白质折叠模型描述了与新生多肽接触的其他成分，但我们的数据表明，HSP70和HSP90家族是主要的分子伴侣，它们与细胞质新生多肽的一般群体相互作用。
Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.