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Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach

机译：通过两杂交分析研究酵母Hsp90分子伴侣系统的蛋白质-蛋白质相互作用：这种方法的潜在用途和局限性

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The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins (“cochaperones”) that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages. To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid–binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast. These “bait” fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD). High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome. Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress. Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 “prey” fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the “relaxed” conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90. A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.
机译：Hsp90分子伴侣循环涉及不同的含Hsp90的多蛋白复合物的顺序组装，随着循环从其早期到后期的进行，与这些复合物相关的辅助蛋白（“伴侣蛋白”）被交换。为了了解2-杂交系统是否可用于探测此Hsp90系统的相互作用，构建了酵母转化体，该转化体表达与4种Hsp90同工型和各种Hsp90系统融合的Gal4p脱氧核糖核酸结合域（BD）酵母的伴侣蛋白。然后将这些“诱饵”融合物与其他表达酵母的几乎所有6000种蛋白质的转化子进行交配，这些蛋白质以与Gal4p激活域（AD）的融合物形式表达。高通量2杂交筛选揭示了Hsp90和Hsp90系统伴侣分子在体内，彼此之间以及与酵母蛋白质组的各种其他蛋白质之间进行稳定相互作用的能力。与大多数分子伴侣缔合的瞬时一致，与Hsp90本身的相互作用总是弱的，并且通常受压力影响。使用Hsp90-BD诱饵融合和AD-Cdc37“猎物”融合中的突变来提供体外数据的体内证实，该体外数据显示Cdc37p与Hsp90的“松弛”构象相互作用，并提供Cdc37p的指示。与Hsp90的任何明显相互作用都需要在其N端​​进行磷酸化。还鉴定了许多潜在的新颖伴侣分子相互作用，为使用其他技术进一步分析这些相互作用提供了框架。

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