篩選分子伴侶複合物Hsc82-Ids2的潛在受質

Abstract

熱休克蛋白90 (Hsp90) 是真核生物中具高度保留性的分子，在許多細胞內分子機制中扮演著重要的角色，如信號傳導和細胞增殖等等。此外，熱休克蛋白90也參與了眾多蛋白質的折疊、裝配與成熟。在其作用的機制當中，需要大量的蛋白因子幫忙，稱之為輔助性熱休克蛋白，這些蛋白會根據其功能參與熱休克蛋白Hsp90的構象循環並調節其活性。先前的研究表示，IME2依賴性信號蛋白(Ids2)可能是Hsp90的輔助性熱休克蛋白，甚至於其在胺基酸148處位點的磷酸化會影響Ids2和Hsp90之間的相互作用，然而，其中的分子機制仍然尚未知曉。因此，我們從和Ids2有交互作用的蛋白質當中篩選，希望能夠發現需依賴Ids2-Hsp90複合物來進行蛋白折疊的潛在受質。於篩選中發現，S-腺苷甲硫氨酸合酶1 (Sam1) 在ids2Δ菌株中，蛋白質表現和酵素功能均會下降，進一步於螢光顯微鏡中發現，Sam1會在細胞中形成蛋白聚集，然而，有趣的是，ids2的磷酸化突變株並不會影響Sam1的蛋白質表現，卻會損害它的功能。除此之外，Ids2和Hsc82以及Sam1三者之間的物理交互作用已經由免疫沉澱法得到證實。結合上述證據，Ids2可能會作為輔助性熱休克蛋白來影響Ids2-Hsc82-Sam1 comeplex的活性。

We have previously found a protein, Ids2 (IME2-dependent signaling protein), de-phosphorylated under CR, and loss of Ids2 leads to CLS extension, mitochondrial defects, and heat intolerant phenotype. The analysis of tandem affinity purification of Ids2 revealed its interaction with Hsp90, including Hsc82 and Hsp82 in yeast. Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone in eukaryotes, which is necessary for folding, maturation, and activity of numerous client proteins. Thus, it plays an essential role in many physiological processes such as signal transduction and cell proliferation. Such chaperone function requires a large group of helper proteins, termed co-chaperones, which participate in the various conformational state of the HSP90 chaperone cycle and regulate its activity. Previous evidence indicated that Ids2 may serve as a co-chaperone of HSP90 and its phosphorylation at residue 148 may affect the interaction between Ids2 and HSP90. However, the molecular mechanism remains elusive. I therefore screened interacting partners of Ids2, hoping to discover potential clients whose protein folding is dependent on the Ids2-HSP90 complex. S-adenosylmethionine synthase 1 (Sam1) was found that both the protein expression and enzymatic function of Sam1 decreased in ids2∆ strains. The further evidence showed that deletion of IDS2 led to foci formation of Sam1; nevertheless, interestingly, ids2 phosphor-mimicking mutation did not influence the protein expression of TAP-tagged Sam1 but did damage the function of it. Besides, the physical interaction was confirmed between Ids2, Hsc82, and Sam1. Together, Ids2 may function as a co-chaperone to affect the activity of the Ids2-Hsc82-Sam1 complex.