Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro
Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro
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DOI：
10.1074/jbc.m208926200
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发表时间：
2002-11-29
影响因子：
4.8
通讯作者：
Vierling, E
中科院分区：
文献类型：
--
作者：
Giese, KC;Vierling, E
小热休克蛋白 (sHSP) 防止其他蛋白质热聚集的能力可能需要 sHSP 寡聚物的拆卸和重新组装。我们通过研究寡聚体稳定性降低的突变体来研究 sHSP 寡聚化变化的作用。 HSP16.6 是蓝藻中的单个 sHSP。集胞藻属。 PCC 6803，L66A 突变导致寡聚物不稳定并降低体外伴侣活性。由于集胞藻的耐热性取决于 HSP16.6（一种在 DeltaClpB1 菌株中增强的表型），因此也可以在体内测定突变的影响。 L66A 会导致严重的耐热性缺陷，这表明 sHSP 的寡聚稳定性是细胞功能所必需的。这一假设得到了 L66A 基因内抑制因子选择的支持，该抑制因子鉴定了稳定 L66A 和野生型 HSP16.6 寡聚体的突变。对寡聚化过度和寡聚化不足的突变体的分析表明，sHSP 必须先分解才能释放底物。此外，抑制突变不仅恢复了 L66A 的体内活性，还改善了体外伴侣缺陷，从而为 sHSP 在细胞耐热性中的伴侣功能提供了第一个直接证据。
The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium. Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a DeltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligoinerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance.