Abstract：
The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
摘要：
胞质铁-硫（Fe-S）簇组装（CIA）途径将Fe-S簇传递给参与基本细胞功能的核和胞质Fe-S蛋白。虽然输送过程是由铁和氧的可用性来调节的，尚不清楚如何CIA组件在不同的蜂窝环境下协调集群转移。这里,我们利用靶向蛋白质组学检测来监测CIA因子和底物，以表征CIA机制。我们发现核苷酸结合蛋白1（NUBP1/NBP35），胞质铁硫组装组件3（CIAO3/NARFL），和CI与核苷酸结合蛋白2（NUBP2/CFD1）相关的底物，CIA支架复合体的组成部分。NUBP2还与CIA靶向复合物（MMS19，CIAO1和CIAO2B）弱相关，表明可能存在高级复合物。补铁或低氧张力后，CIAO3和CIA支架复合物之间的相互作用得到加强，而铁螯合和活性氧削弱了CIAO3与CIA组分的相互作用。我们进一步证明，在Fe-S簇结合中有缺陷的CIAO3突变体无法整合到更高级的复合物中。然而,在与CIA靶向复合物的结合减少的条件下，这些突变体与CIA底物表现出更强的结合，这表明CIAO3和CIA底物可以独立于CIA靶向复合物结合在复合物中。一起,我们的数据表明，CIA成分可能形成代谢产物，其组装受环境线索调节，并且需要在CIAO3中掺入Fe-S簇。这些发现提供了额外的证据，表明CIA通路通过复杂的重组适应细胞环境的变化。
