Pandey Ashutosh K, Pain Jayashree, J Brindha, Dancis Andrew, Pain Debkumar
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ 07103, United States.
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ 07103, United States.
Mitochondrion. 2023 Mar;69:104-115. doi: 10.1016/j.mito.2023.02.006. Epub 2023 Feb 9.
Iron-sulfur (Fe-S) cluster assembly in mitochondria and cytoplasm is essential for cell viability. In the yeast S. cerevisiae, Leu1 [4Fe-4S] is the cytoplasmic isopropylmalate isomerase involved in leucine biosynthesis. Using permeabilized Δleu1 cells and recombinant apo-Leu1, here we show that the [4Fe-4S] cluster assembly on Leu1 can be reconstituted in a physiologic manner requiring both mitochondria and cytoplasm, as judged by conversion of the inactive enzyme to an active form. The mitochondrial contribution to this reconstitution assay is abrogated by inactivating mutations in the mitochondrial ISC (iron-sulfur cluster assembly) machinery components (such as Nfs1 cysteine desulfurase and Ssq1 chaperone) or the mitochondrial exporter Atm1. Likewise, depletion of a CIA (cytoplasmic iron-sulfur protein assembly) component Dre2 leads to impaired Leu1 reconstitution. Mitochondria likely make and export an intermediate, called X-S or (Fe-S), that is needed for cytoplasmic Fe-S cluster biosynthesis. Here we show that once exported, the same intermediate can be used for both [2Fe-2S] and [4Fe-4S] cluster biogenesis in the cytoplasm, with no further requirement of mitochondria. Our data also suggest that the exported intermediate can activate defective/latent CIA components in cytoplasm isolated from nfs1 or Δatm1 mutant cells. These findings may provide a way to isolate X-S or (Fe-S).
线粒体和细胞质中的铁硫（Fe-S）簇组装对于细胞活力至关重要。在酿酒酵母中，Leu1 [4Fe-4S]是参与亮氨酸生物合成的细胞质异丙基苹果酸异构酶。利用透化的Δleu1细胞和重组脱辅基Leu1，我们在此表明，通过将无活性酶转化为活性形式判断，Leu1上的[4Fe-4S]簇组装可以在线粒体和细胞质均需要的生理方式下重建。线粒体对该重建试验的贡献可通过线粒体ISC（铁硫簇组装）机制组分（如Nfs1半胱氨酸脱硫酶和Ssq1伴侣蛋白）或线粒体输出蛋白Atm1中的失活突变而消除。同样，CIA（细胞质铁硫蛋白组装）组分Dre2的缺失导致Leu1重建受损。线粒体可能产生并输出一种称为X-S或（Fe-S）的中间体，这是细胞质Fe-S簇生物合成所必需的。我们在此表明，一旦输出，相同的中间体可用于细胞质中[2Fe-2S]和[4Fe-4S]簇的生物合成，而不再需要线粒体。我们的数据还表明，输出的中间体可激活从nfs1或Δatm1突变细胞分离的细胞质中有缺陷/潜在的CIA组分。这些发现可能提供一种分离X-S或（Fe-S）的方法。
