Highlights d COA6 is a coiled-coil-helix-coiled-coil-helix domain containing protein d COA6 preferentially interacts with SCO1 over SCO2 d COA6 acts as a disulfide reductase of SCO1 and COX2 d COA6 function can be bypassed under hypoxic conditions

In eukaryotes, mitochondria have been hypothesized to generate sulfur species required for tRNA thiolation in the cytosol, although no direct evidence thus far exists. Here we have detected these sulfur species, making use of our observation that isolated yeast cytosol alone is unable to thiolate tRNAs but can do so upon addition of mitochondria. Mitochondria were found to utilize the cysteine desulfurase Nfs1 to produce sulfur-containing species with masses ranging from 700 to 1,100 Da. Mitochondria exported these species via the Atm1 transporter in the inner membrane. Once exported to the cytosol, these sulfur species promoted cytosolic tRNA thiolation with no further requirement of mitochondria. Furthermore, we found that the Isu1/2 scaffolds but not the Ssq1 chaperone of the mitochondrial iron-sulfur cluster machinery were required for cytosolic tRNA thiolation, and thus the sulfur utilization pathway bifurcates at the Isu1/2 site for intra-organellar use in mitochondria or export to the cytosol.

Edited by Ruma Banerjee Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O 2 concentrations to better understand their global iron content. We investigated WT cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin Fe II species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered Fe III ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively coupled plasma mass spectrometer revealed a low-molecular-mass (LMM) Fe II pool consisting of two iron complexes with masses of ϳ500 (major) and ϳ1300 (minor) Da. They appeared to be high-spin Fe II species with mostly oxygen donor ligands, perhaps a few nitrogen donors, and probably no sulfur donors. Surprisingly, the iron content of E. coli and its reactivity with O 2 were remarkably similar to those of mitochondria. In both cases, a "respiratory shield" composed of membrane-bound iron-rich respiratory complexes may protect the LMM Fe II pool from reacting with O 2. When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria.

Blood contains a poorly characterized pool of labile iron called non-transferrin-bound iron (NTBI). In patients with iron-overload diseases such as hemochromatosis, NTBI accumulates in the liver, heart, and other organs. This material is probably nonproteinaceous and low molecular mass (LMM). However, the number, concentration, mass, and chemical composition of NTBI species remain unknown despite decades of effort. Here, solutions of plasma from humans, pigs, horses, and mice were passed through a 10 kDa cutoff membrane, affording flow-through solutions (FTSs) containing ∼1 μM iron. The FTSs were subjected to size-exclusion liquid chromatography at pH 8.5, 6.5, and 4.5. Iron was detected by an online inductively-coupled-plasma mass spectrometer. LC-ICP-MS chromatograms of the FTSs exhibited 2-6 iron-containing species with apparent masses between 400 and 2500 Da. Their approximate concentrations in plasma were 10-8-10-7 M. Not every FTS sample contained every LMM iron species, indicating individual variations. The most reproducible iron species had apparent masses of 400 and 500 Da. Chromatograms of the FTSs from established hemochromatosis patients exhibited no significant differences relative to controls. The peak positions and intensities depended on column pH. Some FTS iron adsorbed onto the column, especially at higher pH. Column-adsorbing-iron coordinated apo-transferrin whereas the more tightly coordinated iron species did not. Ferric citrate standards exhibited LMM iron peaks that were similar to but not the same as those obtained in FTSs. The results indicate that the LMM iron species in healthy blood plasma is not primarily ferric citrate; however, this may be one of many contributing complexes.

Vacuoles play major roles in the trafficking, storage, and homeostasis of metal ions in fungi and plants.