ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells
ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells
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DOI：
10.1046/j.1365-2958.2000.01951.x
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发表时间：
2000-06
影响因子：
3.6
通讯作者：
Jeffrey G. Thomas;F. Baneyx
中科院分区：
文献类型：
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作者：
Jeffrey G. Thomas;F. Baneyx
DnaK–DnaJ–GrpE 和 GroEL–GroES 是大肠杆菌细胞质中特征最好的分子伴侣系统。许多其他蛋白质，包括 ClpA、ClpB、HtpG 和 IbpA/B，在体外充当分子伴侣，但它们在细胞蛋白质折叠中的功能仍不清楚。在这里，我们研究了这些伴侣如何在热激条件下影响新合成的重组蛋白的折叠。我们发现，在 42°C 下，ClpB 或 HtpG 的缺失会导致 preS2-β-半乳糖苷酶聚集增加，preS2-β-半乳糖苷酶是一种融合蛋白，其折叠取决于 DnaK-DnaJ-GrpE，而不是 GroEL-GroES。然而，只有 ΔclpB 突变对同二聚体 Rubisco 和 cMBP 的折叠有害，这两种蛋白质需要 GroEL-GroES 伴侣蛋白才能达到正确的构象。 clpA 或 ibpAB 操纵子中的无效突变不会影响这些模型底物的折叠。 ClpB、HtpG、IbpA/B 或 ClpA 的过表达不会抑制野生型细胞中易于聚集的蛋白 preS2-S'-β-半乳糖苷酶形成包涵体，也不会减轻 dnaJ259、grpE280 或 groES30 突变体中重组蛋白的错误折叠。相比之下，较高水平的 DnaK–DnaJ（而非 GroEL–GroES）可以恢复 ΔclpB 细胞的有效折叠。这些结果表明，ClpB 以及较小程度的 HtpG 参与轻度应激的大肠杆菌细胞中的从头蛋白质折叠，可能是通过扩展 DnaK-DnaJ-GrpE 团队与新合成的多肽相互作用的能力。
DnaK–DnaJ–GrpE and GroEL–GroES are the best‐characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat‐shock conditions. We show that the absence of either ClpB or HtpG at 42°C leads to increased aggregation of preS2‐β‐galactosidase, a fusion protein whose folding depends on DnaK–DnaJ–GrpE, but not GroEL–GroES. However, only the ΔclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL–GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation‐prone protein preS2‐S′‐β‐galactosidase in wild‐type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK–DnaJ, but not GroEL–GroES, restore efficient folding in ΔclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK–DnaJ–GrpE team to interact with newly synthesized polypeptides.