Bcp1蛋白在出芽酵母中核醣體生合成途徑之研究

Bcp1 is a protein involved in ribosome biogenesis in Saccharomyces cerevisiae

陳柏儒(Bo-Ru Chen)

指導教授 ： 羅凱尹

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02147

摘要

真核細胞裡核醣體生合成 (Ribosome biogenesis) 過程中，需要很多非核醣體蛋白幫助，這一類協助核醣體生合成的蛋白稱為輔助蛋白(transacting factors) 。當這些輔助蛋白發生問題時，會導致核醣體生合成的缺失並進而影響蛋白質的生成，使細胞生長受阻，更嚴重的甚至導致細胞死亡。BCP1 是發芽酵母菌 (Saccharomyces cerevisiae) 的一個必需基因 (enssetial gene)，當BCP1有缺失時會導致酵母菌死亡。前人研究指出Bcp1與60S核醣體蛋白Rpl23 (large subunit ribosomal protein 23) 之間有交互作用，此外也跟酸肌醇磷酸激酶蛋白Mss4 (Phosphatidylinositol-4-phosphate 5-kinase) 的出核運輸有關，因此Bcp1應為一個輔助蛋白，其在核醣體生合成過程中可能的功能與角色是本研究的主要研究方向。
在本研究首先重複前人的研究成果，證明Bcp1蛋白的缺失會造成大核醣體無法被運輸至細胞質而累積在細胞核裡，並造成60S生成的缺陷。
本研究並針對Bcp1蛋白本身可能具有特定功能胺基酸序列進行刪除或突變，製作成bcp1Δn40、bcp1Δc、bcp1Δnls、bcp1 nes突變株，研究這些特定胺基酸對於Bcp1蛋白功能上的影響。結果發現這些胺基酸序列的改變都會導致60S 核醣體生合成受損，但是我們意外的發現在生長測試結果中BCP1Δnls突變株卻能夠存活，其餘的突變株皆會造成細胞死亡，因此，我們認為Bcp1蛋白在細胞質中可能有重要的功能。
接著，本研究針對BCP1與export factors (NMD3, ECM1, ARX1, MEX67, MTR2) 之間基因關聯性的分析，結果顯示BCP1和export factors有基因關聯性並且當同時產生缺失時會產生synthetic sick現象，而且60S核醣體生成變的更差。我們想藉由大量表現export factors蛋白來修補Bcp1蛋白失活造成的生長缺陷，但是沒有效果，因此，我們認為Bcp1缺失時可能影響其他途徑，間接影響60S出核運輸。
為了找尋Bcp1的另一個參與的途徑，我們針對能與Bcp1蛋白結合的Rpl23蛋白著手研究，結果顯示Rpl23可以修補Bcp蛋白失活所導致細胞死亡的現象，是Bcp1蛋白的高量抑制子 (high copy suppressor) ，且在分析核醣體生合成圖譜時，亦觀察到60S合成的缺失有被修復的現象。
本研究又針對結合於Rpl23上，防止未成熟的60S核醣體與40S結合的Tif6蛋白進行研究。本研究發現在Bcp1蛋白失活的情況下，會造成Tif6留在細胞質的比例增加。而且Bcp1蛋白失活也會導致在核醣體成熟過程中在Tif6下游被釋出的Nmd3蛋白在細胞質分布的比例上升。接著本研究針對協助Tif6從60S釋出的蛋白Sdo1以及Efl1進行研究，發現在bcp1ts突變株中Sdo1以及Efl1表現狀況與野生珠沒有差異。
根據以上結果，Bcp1參與60S核醣體的生合成，且其缺失可以被Rpl23抑制。Bcp1缺失會造成Tif6及Nmd3的位置改變，進而影響其功能，但其機制為何，尚待進一步的實驗說明。

關鍵字

Bcp1蛋白 ； 核醣體生合成 ； 出核運輸蛋白 ； Rpl23 ； 高量抑制子 ； Tif6

並列摘要

BCP1 is an essential gene in Saccharomyces cerevisiae. According to the previous research, Bcp1 is involved in the export of Mss4. Furthermore, when Bcp1 has defects, 60S subunits can't be exported from the nucleus. It implies that Bcp1 is also involved in 60S biogenesis.
First, we mutated or deleted the function domains of Bcp1 and made mutant constructs: bcp1Δn40、bcp1Δc、bcp1Δnls、bcp1nes.　All these mutants showed 60S ribosomal biogenesis defects. Except bcp1Δnls all other mutatnts can’t complement the growth of bcp1ts at nonpermissive temperature. .
Then we analyzed the genetic interaction between BCP1 and 60S export factors ( NMD3, ECM1, ARX1, MEX67, MTR2 ). We found the double mutants grew much sicker than those single mutants, which means Bcp1 has tight functional connection with these export factors. However, overexpression of any of the export factors can’t rescue bcp1ts and vice versa. Therefore, Bcp1 may involve in another pathway. The accumulation of 60S might be from the secondary effects of the block of primary pathway.
We tried to test if Bcp1 showed any functional connection with Rpl23, which shows physical interaction with Bcp1. Interesting, we found overexpression of Rpl23 could rescue bcp1ts growth at nonpermissive temperature. Rpl23 protein levels were also highly elevated when Bcp1 function was inactivated.
To futher dissect the mechanism about why Rpl23 could rescue bcp1ts, we tested Tif6. Tif6 is a 60S transacting factor which binds at Rpl23 at joining face. It escorts with 60S subunits to the cytoplasm and needs to be released before ribosome maturation. Failure of Tif6 release blocks 40S association with 60S and results in cell lethality. Interesting, Tif6 was mislocalized when bcp1 was inactivatded. However, mutations of Bcp1 does not impact Tif6 release but stability.
According to our studies, Bcp1 plays an important role in 60S ribosome biogenesis. Its function is tightly connect to Tif6 and Rpl23. However, the mechanism is still unclear and futher studies are required.

並列關鍵字

Bcp1 ； ribosome biogenesis ； export factor ； Rpl23 ； high copy suppressor ； Tif6

參考文獻

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