Yeast Mitochondria Import Aqueous Fe^II and, When Activated for Iron–Sulfur Cluster Assembly, Export or Release Low-Molecular-Mass Iron and Also Export Iron That Incorporates into Cytosolic Proteins

Abstract

Iron–sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for cytosolic ISC assembly. This species, called X–S or (Fe–S)int, has not been directly detected. Here, an assay was developed in which mitochondria were isolated from 57Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous 54FeII in the buffer declined upon exposure to intact 57Fe-enriched mitochondria. Some 54Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in 54Fe and 57Fe, suggesting that the added 54Fe entered a pre-existing pool of 57Fe, which was also the source of the exported species. When 54Fe-loaded 57Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when 54Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with 57Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.

Sterol-targeted laboratory evolution allows the isolation of thermotolerant and respiratory-competent clones of the industrial yeast Saccharomyces cerevisiae

“…The results con rm the interplay between mitochondrial respiration and sterol biosynthesis, as has been previously evidenced [74]. Mitochondria harbours the heme and iron-sulfur (Fe-S) 2 cluster synthesis [75], which is essential for the activity of some sterol biosynthesis enzymes, and determinant in the oxidant radical formation [76]. In turn, the accumulation of toxic methyl sterols by, for instant, azoles treatment or loss of Erg25 or Erg27 activity, results in a rise in mitochondrial oxidants, a decrease in Fe-S production, and nally a loss of mitochondrial DNA [77,78].…”

Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a β-Lactam to Gram-Negative Bacterial Pathogens

“…To measure siderophore transport, we treated S Tm with 57 Fe­(III)-loaded Ent or TC and monitored 57 Fe uptake by quantifying cell-associated 57 Fe by using inductively coupled plasma mass spectrometry (ICP-MS). As a stable, low abundance isotope (2.12% abundance vs 91.8% for 56 Fe), 57 Fe can effectively be used as a biological tracer; we and others have monitored the ratios of 57 Fe to other isotopes of Fe as an approach to track different Fe sources. − We chose to measure uptake in S Tm as a case study because this gastrointestinal pathogen produces and utilizes Ent and employs both FepA and IroN to transport it across the OM, and we recently demonstrated that Ent-based SACs are highly active against S Tm . Additionally, S Tm only produces the catecholate siderophores Ent and salmochelin.…”