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.2022 Nov 1;14(11):mfac080.
doi: 10.1093/mtomcs/mfac080.

Mössbauer-based molecular-level decomposition of the Saccharomyces cerevisiae ironome, and preliminary characterization of isolated nuclei

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Mössbauer-based molecular-level decomposition of the Saccharomyces cerevisiae ironome, and preliminary characterization of isolated nuclei

Paul A Lindahl et al. Metallomics..

Abstract

One hundred proteins in Saccharomyces cerevisiae are known to contain iron. These proteins are found mainly in mitochondria, cytosol, nuclei, endoplasmic reticula, and vacuoles. Cells also contain non-proteinaceous low-molecular-mass labile iron pools (LFePs). How each molecular iron species interacts on the cellular or systems' level is underdeveloped as doing so would require considering the entire iron content of the cell-the ironome. In this paper, Mössbauer (MB) spectroscopy was used to probe the ironome of yeast. MB spectra of whole cells and isolated organelles were predicted by summing the spectral contribution of each iron-containing species in the cell. Simulations required input from published proteomics and microscopy data, as well as from previous spectroscopic and redox characterization of individual iron-containing proteins. Composite simulations were compared to experimentally determined spectra. Simulated MB spectra of non-proteinaceous iron pools in the cell were assumed to account for major differences between simulated and experimental spectra of whole cells and isolated mitochondria and vacuoles. Nuclei were predicted to contain ∼30 μM iron, mostly in the form of [Fe4S4] clusters. This was experimentally confirmed by isolating nuclei from 57Fe-enriched cells and obtaining the first MB spectra of the organelle. This study provides the first semi-quantitative estimate of all concentrations of iron-containing proteins and non-proteinaceous species in yeast, as well as a novel approach to spectroscopically characterizing LFePs.

Keywords: cytosol; endoplasmic reticulum; iron–sulfur clusters; labile iron pool; mitochondria; nucleus isolation; vacuoles.

© The Author(s) 2022. Published by Oxford University Press.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Graphical Abstract
Graphical Abstract
Lindahl and Vali consider all iron-containing proteins in a yeast cell in simulating the expected Mössbauer spectrum of whole yeast cells.
Fig. 1
Fig. 1
Pie-chart distributions of iron in each compartment of a yeast cell. Charts on the left indicate the major proteins in each considered compartment, given as a percentage of iron contribution. Charts on the right show the percentage of different types of iron centers in the compartment, again as a percentage of iron contribution.
Fig. 2
Fig. 2
Simulated Mössbauer spectra of nuclei fromS. cerevisiae. Temperatures of 4.2–5 K and parallel applied fields of ∼0.05 T were assumed (same for all figures). Individual spectra for each iron-containing protein in the nucleus, as indicated, were simulated and summed to generate the predicted spectrum (bottom) of isolated nuclei.
Fig. 3
Fig. 3
Simulated Mössbauer spectra of each cellular compartment and whole Fsa and Rsa yeast cells. Spectra were generated by summing the spectra from each of the five cellular compartments, weighted by the fraction of cellular iron in each compartment.
Fig. 4
Fig. 4
Simulated Mössbauer spectra of whole yeast cells with non-proteinaceous iron included. Simulations of: non-proteinaceous non-heme high-spin FeII (blue line); non-proteinaceous high-spin vacuolar FeIII (red); iron-replete fermenting whole cells (upper black); and respiring (Rsa) whole cells (lower black).
Fig. 5
Fig. 5
Characterization of yeast nuclei.A, Averaged Mössbauer spectrum of isolated nuclei from growth media containing 1 or 40 μM Fe. Solid lines are simulations of the CD (red), NHHS FeII (green), and NHHS FeIII (teal).B, X-band EPR spectrum (5K, modulation amplitude—10 G, modulation frequency—100 kHz, power— 0.2 mW, frequency—9.36 GHz, and sweep time—300 s) of nuclei isolated from cells grown with 1 μM iron.C, Western blot of whole-cell (WC), lysate (Lys), and nuclei (N) against various antibody markers for CPY (vacuoles), Kar2 (ER), PGK (cytosol), Nop1 (nuclei), and porin (mitochondria). D,65Cu-detected chromatograms of flow-through solution from soluble nuclear extracts from three independent preparations collected using LC-ICP–MS.
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