Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol
Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol
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DOI：
10.1046/j.1365-2958.2001.02383.x
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发表时间：
2001-04-01
影响因子：
3.6
通讯作者：
Bukau, B
中科院分区：
文献类型：
--
作者：
Tomoyasu, T;Mogk, A;Bukau, B
我们研究了分子伴侣和蛋白酶在大肠杆菌胞质溶胶蛋白质质量控制中的作用。在Delta rpoH突变体中，缺乏热休克转录因子，因此除了GroEL和触发因子外，所有主要的胞质蛋白酶和伴侣蛋白水平都很低，总蛋白的5-10%和20-30%分别在30 ℃和42 ℃聚集。聚集体含有350-400种蛋白质种类，其中93种通过质谱鉴定。聚集的蛋白质种类在两个温度下是相似的，表明热不稳定蛋白质需要折叠辅助分子伴侣已经在30 degreesC，并表现出强烈的重叠与以前确定的DnaK底物。过量生产的DnaK系统，或低水平生产的DnaK系统和ClpB，防止聚集，并提供耐热性三角洲rpoH突变体，这些分子伴侣蛋白在蛋白质质量控制和应力生存的关键作用。在rpoH（+）细胞中，DnaK耗竭在30 ℃下不导致蛋白质聚集，这可能是高水平蛋白酶的结果，因此表明DnaK不是错误折叠蛋白质的蛋白水解的先决条件。在DnaK缺失的细胞中，Lon是降解错误折叠蛋白质的最有效的蛋白酶。在42 ℃时，ClpXP和Lon对于具有低DnaK水平的细胞的活力变得至关重要，这表明蛋白酶和DnaK系统的协同作用，这对于42 ℃下的细胞生长至关重要。
We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In Delta rpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degreesC and 42 degreesC respectively. The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degreesC, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to Delta rpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH(+) cells, DnaK depletion did not lead to protein aggregation at 30 degreesC, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells. At 42 degreesC, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degreesC.