Chromatographic detection of low-molecular-mass metal complexes in the cytosol of <i>Saccharomyces cerevisiae</i>

Nguyen, Trang Quynh; Kim, Joshua E.; Brawley, Hayley N.; Lindahl, Paul A.

doi:10.1039/c9mt00312f

Cited by 16 publications

(25 citation statements)

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“…We previously reported that labile LMM metal complexes partially adsorbed onto, and desorbed from, the SEC column [ 22 , 23 ]. Metal ions likely participated in secondary ionic interactions with basic groups on the solid support such as carboxylates [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

“…This suggested that PPX chelated the LMM metal species in E. coli cytosol. We performed similar experiments previously to evaluate whether LMM metal species in the cytosol of S. cerevisiae were coordinated by polyphosphate ions [ 22 ]. At that time, we had difficulty interpreting our results (because metal polyphosphate complexes were not expected to be in the cytosol), but they can now be explained by assuming that PPX chelated metals from LMM metal complexes in the cytosol; PPX-sensitive species in the cytosol of S. cerevisiae probably do not coordinate polyphosphate ions.…”

Section: Chromatographic Behavior Of Iron and Zinc Standards Reflected The M–l Binding Strength Of The Complexmentioning

confidence: 99%

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Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

Brawley

Lindahl

2021

J Biol Inorg Chem

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Labile low-molecular-mass (LMM) transition metal complexes play essential roles in metal ion trafficking, regulation, and signalling in biological systems, yet their chemical identities remain largely unknown due to their rapid ligand-exchange rates and weak M–L bonds. Here, an Escherichia coli cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of the metal ions from these complexes with a SEC column was minimized by pre-loading the column with 67ZnSO4 and then monitoring 66Zn and other metals by inductively coupled plasma mass spectrometry (ICP-MS) when investigating cytosolic ultrafiltration flow-through-solutions (FTSs). Endogenous cytosolic salts suppressed ESI-MS signals, making the detection of metal complexes difficult. FTSs contained ca. 80 µM Fe, 15 µM Ni, 13 µM Zn, 10 µM Cu, and 1.4 µM Mn (after correcting for dilution during cytosol isolation). FTSs exhibited 2–5 Fe, at least 2 Ni, 2–5 Zn, 2–4 Cu, and at least 2 Mn species with apparent masses between 300 and 5000 Da. Fe(ATP), Fe(GSH), and Zn(GSH) standards were passed through the column to assess their presence in FTS. Major LMM sulfur- and phosphorus-containing species were identified. These included reduced and oxidized glutathione, methionine, cysteine, orthophosphate, and common mono- and di-nucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the labile metal pools in E. coli is sensitive to the concentration of nutrient metals.

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Section: Resultsmentioning

confidence: 99%

Section: Chromatographic Behavior Of Iron and Zinc Standards Reflected The M–l Binding Strength Of The Complexmentioning

confidence: 99%

Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

Brawley

Lindahl

2021

J Biol Inorg Chem

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“…The cytosol was isolated from fermenting cells (pH 5) and filtered through a 10-kDa cutoff membrane as described ( 55 ). Filtered FTS was injected into a treated size-exclusion (SEC) Superdex Peptide 10/300 Gl column (GE Life Sciences) connected to an Agilent 1260 Bioinert quaternary pump (G5611A) and Agilent 7700x ICP-MS.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot was completed using the whole-cell extract as described ( 12 , 55 ). An anti-Fet3 antibody from rabbit was used in a 1:1000 dilution.…”

Section: Methodsmentioning

confidence: 99%

Mössbauer and LC-ICP-MS investigation of iron trafficking between vacuoles and mitochondria in vma2Δ Saccharomyces cerevisiae

Kim

Vali

Nguyen

et al. 2021

Journal of Biological Chemistry

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Vacuoles are acidic organelles that store Fe III polyphosphate, participate in iron homeostasis, and have been proposed to deliver iron to mitochondria for iron–sulfur cluster (ISC) and heme biosynthesis. Vma2Δ cells have dysfunctional V-ATPases, rendering their vacuoles nonacidic. These cells have mitochondria that are iron-dysregulated, suggesting disruption of a putative vacuole-to-mitochondria iron trafficking pathway. To investigate this potential pathway, we examined the iron content of a vma2Δ mutant derived from W303 cells using Mössbauer and EPR spectroscopies and liquid chromatography interfaced with inductively-coupled-plasma mass spectrometry. Relative to WT cells, vma2Δ cells contained WT concentrations of iron but nonheme Fe II dominated the iron content of fermenting and respiring vma2Δ cells, indicating that the vacuolar Fe III ions present in WT cells had been reduced. However, vma2Δ cells synthesized WT levels of ISCs/hemes and had normal aconitase activity. The iron content of vma2Δ mitochondria was similar to WT, all suggesting that iron delivery to mitochondria was not disrupted. Chromatograms of cytosolic flow–through solutions exhibited iron species with apparent masses of 600 and 800 Da for WT and vma2∆ , respectively. Mutant cells contained high copper concentrations and high concentrations of a species assigned to metallothionein, indicating copper dysregulation. vma2Δ cells from previously studied strain BY4741 exhibited iron-associated properties more consistent with prior studies, suggesting subtle strain differences. Vacuoles with functional V-ATPases appear unnecessary in W303 cells for iron to enter mitochondria and be used in ISC/heme biosynthesis; thus, there appears to be no direct or dedicated vacuole-to-mitochondria iron trafficking pathway. The vma2Δ phenotype may arise from alterations in trafficking of iron directly from cytosol to mitochondria.

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“…None of the above interpretations and conclusions could have been reached without specifically designed experimental setups and analytical methods, and accurate and extensive quantitative data. Attempts at any comprehensive understanding of metal homeostasis cannot avoid such a depth of analysis as witnessed by studies in the minimally autonomous, i.e., able to grow in isolation on a single substrate, and model eukaryote Saccharomyces cerevisiae [ 132 , 133 , 134 ]. However, integration of the data available for this supposedly simple organism requires more than 150 variables and tens of reactions influencing them [ 135 , 136 ].…”

Section: What May Be the Molecular Basis Of Specificity For Cellumentioning

confidence: 99%

Cellular Dynamics of Transition Metal Exchange on Proteins: A Challenge but a Bonanza for Coordination Chemistry

Moulis

2020

Biomolecules

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Transition metals interact with a large proportion of the proteome in all forms of life, and they play mandatory and irreplaceable roles. The dynamics of ligand binding to ions of transition metals falls within the realm of Coordination Chemistry, and it provides the basic principles controlling traffic, regulation, and use of metals in cells. Yet, the cellular environment stands out against the conditions prevailing in the test tube when studying metal ions and their interactions with various ligands. Indeed, the complex and often changing cellular environment stimulates fast metal–ligand exchange that mostly escapes presently available probing methods. Reducing the complexity of the problem with purified proteins or in model organisms, although useful, is not free from pitfalls and misleading results. These problems arise mainly from the absence of the biosynthetic machinery and accessory proteins or chaperones dealing with metal / metal groups in cells. Even cells struggle with metal selectivity, as they do not have a metal-directed quality control system for metalloproteins, and serendipitous metal binding is probably not exceptional. The issue of metal exchange in biology is reviewed with particular reference to iron and illustrating examples in patho-physiology, regulation, nutrition, and toxicity.

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Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae

Abstract: Labile metal pools in the cytosol of yeast, including those of iron, copper, zinc, and manganese, can be detected and characterized using size-exclusion chromatography with online ICP-MS.

Cited by 16 publications

References 57 publications

Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

Mössbauer and LC-ICP-MS investigation of iron trafficking between vacuoles and mitochondria in vma2Δ Saccharomyces cerevisiae

Cellular Dynamics of Transition Metal Exchange on Proteins: A Challenge but a Bonanza for Coordination Chemistry

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