Stimulation of the ATPase activity of the yeast mitochondrial ABC transporter Atm1p by thiol compounds
Stimulation of the ATPase activity of the yeast mitochondrial ABC transporter Atm1p by thiol compounds
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DOI：
10.1080/09687860500473630
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发表时间：
2006-03-01
影响因子：
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通讯作者：
Lill, Roland
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文献类型：
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作者：
Kuhnke, Grit;Neumann, Karina;Lill, Roland
线粒体内膜上的三磷酸腺苷结合盒(ABC)转运体Atm1p在胞质/核铁硫蛋白的生物发生和细胞铁稳态中起着至关重要的作用。由于线粒体铁-硫簇(ISC)组装机械的功能也是这两个过程所必需的，因此Atm1p被认为将这一途径的未知产物转移到细胞质中。为了更好地了解Atm1p的功能，我们在体外对Atm1p进行了详细的研究。利用大肠杆菌表达系统对Atm1p进行纯化。洗涤剂溶解的蛋白质显示出稳定的ATPase活性。将Atm1p重组到蛋白脂质体中，使我们能够确定ATPase的生化特性，如：(1)过渡态类似物钒酸盐对ATPase的强烈抑制；(2)Km值为0.1 mm；(3)ATPase的周转次数为127分钟(-1)。ABC转运蛋白的ATPase活性通常受其特定底物的刺激。我们利用这一性质定义了由Atm1p传输的衬底的化学性质。尽管Atm1p不是一个通用的多肽转运蛋白，如酵母Mdl1p或哺乳动物TAP，但含有Atm1p的蛋白脂质体对ATPase的水解率可以被含有硫醇的化合物特异性地提高3-5倍，特别是多肽中半胱氨酸硫醇的微摩尔浓度。我们推测，Atm1p的生理底物可能在多肽环境中含有多个巯基。
The ATP binding cassette (ABC) transporter Atm1p of the mitochondrial inner membrane performs crucial roles in both the biogenesis of cytosolic/nuclear iron-sulfur proteins and cellular iron homeostasis. Since the function of the mitochondrial iron-sulfur cluster (ISC) assembly machinery is also required for these two processes, Atm1p is thought to translocate a still unknown product of this pathway to the cytosol. Here, we provide a detailed in vitro characterization of Atm1p in order to better understand its function. Atm1p was purified using an expression system in E. coli. The detergent-solubilised protein exhibits a stable ATPase activity. Reconstitution of Atm1p into proteoliposomes allowed us to determine the biochemical characteristics of the ATPase such as: (i) the strong inhibition by the transition state analogue vanadate, (ii) a Km value of 0.1 mM, and (iii) a turnover number of 127 min(-1). The ATPase activity of ABC transporters is generally stimulated by their specific substrate. We used this property to define the chemical properties of the substrate transported by Atm1p. ATPase hydrolysis by Atm1p-containing proteoliposomes was specifically increased 3-5-fold by thiol-containing compounds, in particular by micromolar concentrations of cysteine thiol groups in peptides, even though Atm1p is not a general peptide transporter such as yeast Mdl1p or mammalian TAP which share sequence similarity with Atm1p. We speculate that the physiological substrate of Atm1p may contain multiple sulfhydryl groups in a peptidic environment.