A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo
A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo
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DOI：
10.1073/pnas.0509583102
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发表时间：
2005-12-27
影响因子：
11.1
通讯作者：
Beckwith, J
中科院分区：
文献类型：
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作者：
Huber, D;Cha, MI;Beckwith, J
大肠杆菌硫氧还蛋白通常是一种参与二硫键还原的细胞质蛋白。然而，当硫氧还蛋白附着在一个共译信号序列上时，它可以被转运到外质。当输出到外周质时，它可以部分取代DsbA促进二硫键形成的活性。相反，当硫氧还蛋白融合到翻译后信号序列时，很少会出现在外周质中。我们认为这种翻译后输出的缺失是由于细胞质中硫氧还蛋白的快速折叠。我们通过将硫氧还蛋白融合到翻译后的信号序列中，并选择硫氧还蛋白输出到细胞质外周的突变体，在那里硫氧还蛋白可以取代DsbA，从而寻找能延缓其在细胞质中折叠的突变体。获得的突变体集合代表了蛋白质中有限数量的氨基酸变化。对纯化突变蛋白的体外研究表明，除了一个突变蛋白外，其他突变蛋白在蛋白质折叠的动力学和热力学方面都存在缺陷。我们提出，硫氧还蛋白突变蛋白在细胞质中较慢的折叠允许它们通过翻译后途径输出。我们讨论了这类突变体对硫氧还蛋白折叠途径及其输出机制的影响。特别是，发现允许蛋白质易位的折叠突变体改变了蛋白质C端的氨基酸，这表明硫氧还蛋白在翻译过程中折叠的程度必须受到严格限制。
Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.