人類粒線體第一蛋白質複合體中NDUFS7次單元擁有粒線體與細胞核兩種不同的分佈

Human mitochondrial complex I NDUFS7 subunit has a dual distribution both in mitochondria and nuclei

周定芳(Ting-Fang Chou)

指導教授 ： 高茂傑

國立清華大學/生命科學院/分子醫學研究所/碩士(2009年)

https://doi.org/10.6843/NTHU.2009.00252

摘要

人類粒線體第一蛋白質複合體中的NDUFS7（NADH dehydrogenase ubiquinone Fe-S protein 7）在演化上具有高度保留的特性，並在氧化磷酸化系統中（oxidative phosphorylation system, OXPHOS）扮演重要的角色。NDUFS7含有一個[4Fe-4S]的鐵硫中心（iron-sulfur cluster N2, tetranuclear），其功能為擔任第一蛋白質複合體中電子傳遞的最後電子接受者。於臨床研究中發現三種NDUFS7的突變與Leigh症候群（Leigh syndrome, LS）有關。NDUFS7是由細胞核基因組所轉錄，於細胞質轉譯成蛋白質後再進入粒線體，並組裝於第一蛋白質複合體的親水區塊。在T-REx-293細胞內，利用核醣核酸干擾技術(RNAi)來抑制NDUFS7的表現，而NDUFS7表現下降會導致細胞在含有乳糖的培養基中生長緩慢以及細胞中H2O2上升等表型。此結果顯示NDUFS7在粒線體能量代謝及細胞的生長扮演重要角色。多數表現在粒線體基質的蛋白質是在細胞質中合成，且藉由粒線體膜上的TIM/TOM蛋白質複合體辨認本身攜帶的粒線體標的訊號（mitochondrial targeting signal, MTS）來運輸。經預測軟體分析，NDUFS7的N端前29個胺基酸可能為MTS的所在。在本次實驗中，我們建構出刪除不同片段大小的NDUFS7與綠螢光蛋白（EGFP）融合的質體，來觀察其在T-REx-293細胞內的表現位置。 我們發現NDUFS7前60個胺基酸的片段即具有良好運送EGFP至粒線體的能力,顯示MTS位於此蛋白的N端。 接著我們藉由定點突變的方法來研究鹼性與疏水性胺基酸對於此MTS的功能影響，發現這兩類胺基酸對於NDUFS7 MTS的運輸能力皆很重要。另外，我們也定義出NDUFS7的C端區域含有一段有效的細胞核輸入訊號（nuclear localization signal, NLS）以及細胞核運出訊號（nuclear export signal, NES）。由這些結果可證明NDUFS7在細胞中擁有粒線體與細胞核兩種不同的分佈。

關鍵字

粒線體 ； 標的訊號 ； 免疫螢光染色

並列摘要

Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7), one of the most conserved core subunits of mitochondrial complex I, plays an important role in the oxidative phosphorylation system (OXPHOS). This protein binds one iron-sulfur cluster N2 (tetranuclear) which is the terminal redox center in the electron transport process of complex I. Three types of mutations in this subunit have been associated with Leigh syndrome (LS). NDUFS7 protein is encoded by nuclear genome and incorporated in the peripheral segment of complex I facing the mitochondrial matrix. The NDUFS7 was suppressed in human T-REx-293 cells using the RNA interference (RNAi) technology. The reduction in the NDUFS7 expression caused a slow growth rate in galactose containing medium and increased H2O2 generation. These results indicated that NDUFS7 may play an important role in cell energy production and survival. Most mitochondrial matrix proteins are synthesized in the cytoplasm and imported into mitochondria by TIM/TOM complexes recognizing the mitochondrial targeting sequences (MTSs). Using predication softwares, the N-terminal fragment was suggested to be the MTS of NDUFS7. In this study, we fused NDUFS7 containing N-terminal deletions, C-terminal deletions or different portions of the protein to enhanced green fluorescent protein (EGFP), and used these obtained constructs to study their intracellular localization in T-REx-293 cells. We found that the chimeric NDUFS71-60-EGFP was colocalized with mitochondria, demonstrating that this N-terminal fragment contained an effective MTS. We then used site-directed mutagenesis analyses to study the role of basic and hydrophobic residues in the first 60 amino acids of NDUFS7. These results suggested that both two types of amino acids are important for the mitochondrial import of the MTS in NDUFS7. In addition, we also demonstrated that there is a nuclear localization signal (NLS) and a nuclear export signal (NES) located in the C-terminus of NDUFS7. These results suggested that NDUFS7 has a dual distribution both in mitochondria and nuclei.

並列關鍵字

mitochondria ； signal peptide ； immnofluorescence

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