The lack of synchronization between iron uptake and cell growth leads to iron overload in Saccharomyces cerevisiae during post-exponential growth modes

Abstract

Fermenting cells growing exponentially on rich (YPAD) medium underwent a transition to a slow-growing state as glucose levels declined and their metabolism shifted to respiration. During exponential growth, Fe import and cell-growth rates were matched, affording an approximately invariant cellular Fe concentration. During the transition period, the high-affinity Fe import rate declined slower than the cell-growth rate declined, causing Fe to accumulate, initially as Fe(III) oxyhydroxide nanoparticles but eventually as mitochondrial and vacuolar Fe. Once the cells had reached slow-growth mode, Fe import and cell-growth rates were again matched, and the cellular Fe concentration was again approximately invariant. Fermenting cells grown on minimal medium (MM) grew more slowly during the exponential phase and underwent a transition to a true stationary state as glucose levels declined. The Fe concentration of MM cells that just entered the stationary state was similar to that of YPAD cells, but MM cells continued to accumulate Fe in the stationary state. Fe initially accumulated as nanoparticles and high-spin Fe(II) species, but vacuolar Fe(III) also eventually accumulated. Surprisingly, Fe-packed 5-day-old MM cells suffered no more reactive oxygen species (ROS) damage than younger cells, suggesting that the Fe concentration alone does not accurately predict the extent of ROS damage. The mode and rate of growth at the time of harvesting dramatically affected cellular Fe content. A mathematical model of Fe metabolism in a growing cell was developed. The model included the import of Fe via a regulated high-affinity pathway and an unregulated low-affinity pathway. The import of Fe from the cytosol to vacuoles and mitochondria and nanoparticle formation were also included. The model captured essential trafficking behavior, demonstrating that cells regulate Fe import in accordance with their overall growth rate and that they misregulate Fe import when nanoparticles accumulate. The lack of regulation of Fe in yeast is perhaps unique compared to the tight regulation of other cellular metabolites. This phenomenon likely derives from the unique chemistry associated with Fe nanoparticle formation.

Figure 1

Chronological profile of nutrient, growth and Fe-associated parameters for cells grown in batch culture on YPAD for 5 days. A, full plots; B, zoom of 4 – 20 hr; C, 0 – 14 hr for a follow-up experiment. Data include OD₆₀₀ (circles), cellular Fe concentration (squares), glucose concentration (triangles) and Fet3p expression (diamonds). Orange vertical lines demarcate exponential and transitionary modes; purple lines demarcate transitionary and slow-growing modes. Plotted OD₆₀₀ and iron(glucose) concentrations in A and B were the average of two(three) independent experiments. Bars indicate standard deviation.

Figure 2

Chronological profile of nutrient, growth and Fe-associated parameters for cells grown in batch culture on MM for 5 days. A, full plots; B, zoom of 5 – 35 hr; C, 0 – 14 hr for a follow-up experiment. Data symbols and lines are the same as in Figure 1. Reported OD₆₀₀ and iron(glucose) concentrations in A and B were the average of two(three) independent experiments. Bars indicate standard deviation.

Figure 3

5 K 0.04 T Mösbauer spectra of whole yeast cells grown on YPAD and harvested at various time points. A, early transitionary phase (OD 2.1); B, late transitionary phase (OD 3.2); C, B-minus-A difference spectrum; D, post-diauxic growth phase (OD 8.0); E, D-minus-B difference spectrum. Red lines simulate collectively the spectral features associated with HS Fe^III, NHHS Fe^II, HS Fe^II heme, CD, and Fe^III nanoparticles. Brown, purple, yellow, green and blue lines are simulations of HS Fe^III, NHHS Fe^II, HS Fe^II heme, the central doublet, and Fe^III nanoparticles, respectively. The field was applied parallel to γ-rays.

Figure 4

Mössbauer spectra of whole yeast cells grown on MM and harvested at various time points. For A – E, the temperature was 5 K and a 40 mT field was applied parallel to γ-rays. For F, the temperature was 4.2 K and a 6 T field was applied perpendicular to the γ-rays. A, early exponential phase (OD 0.2); B, exponential phase (OD 1.2); C, transitionary phase (OD 1.8); D, C-minus-B difference spectrum (the spectral intensity of B was doubled and then subtracted from C) E, stationary phase (OD 1.8, 5-day-grown); F, an equivalent sample of E (OD 1.7, 5-day-grown). Red lines are composite simulations that include contributions from HS Fe^III, NHHS Fe^II, HS Fe^II heme, CD, and Fe^III nanoparticles. Blue and green lines simulate contributions from Fe^III nanoparticles and HS Fe^II, respectively. The brown line simulates the HS Fe^III species using S = 5/2, D = 0.15 cm⁻¹, E/D = 0.21, A₀/g_N·β _N = −233 kG, δ = 0.55 mm/s, ΔE_Q = 0.42 mm/s, and η = 1.3.

Figure 5

X-band EPR spectra of 5-day-grown cells. A, grown on MM with 40 μM Fe; B, grown on MM with 400 μM Fe. Black, 10 K; blue, 30 K; red, 80 K. Spectra were recorded at 0.05 mW microwave power, 9.64 GHz frequency and 10 Gauss modulation amplitude, and then have been adjusted vertically so that the g = 4.3 resonances are aligned. Spectral intensities in A and B were multiplied by absolute temperature while those in A were additionally multiplied by 5 for presentation purposes.

Figure 6

Mösbauer spectra of MM-grown yeast cells at stationary phase. A, 400 μM Fe-containing MM-grown cells at stationary phase (OD 2.2, 5-day-grown). The red line simulates the contributions from HS Fe^III, Fe^III nanoparticles and the CD. The blue line simulates the ICS-mutant-type nanoparticles. B, same as A but after subtracting the red-line simulation. For A and B, the temperature was 5 K and a 40 mT field was applied parallel to γ-rays. C, same as A but at 6 T (transverse field) and 4.2 K; D, same as in C but the HS Fe^III component has been subtracted. Dotted line is the equivalent MB spectrum of isolated mitochondria from Aft1-1^up cells. The brown line simulates the HS Fe^III contribution.

Figure 7

Oxyblot and Western blot against Sod2p of whole cells and mitochondria harvested at different conditions. A, Oxyblot of whole-cell lysates (left panel) and mitochondrial extracts (right panel). Medium and days of growth are displayed on the top of blots. Overall band intensities (below blots) are indicated as percentages relative to YPAD/1 day (left) or MM/1 day (right) blots. B, Oxyblot time-course of whole cell lysates grown on MM. Band intensities (below blots) are indicated as percentages relative to that of MM/24 hr blot. The intensity at ~29 kDa between lanes 3–4 is an artifact. C, Western blot against Sod2p for YPAD and MM cells grown for different times (same MM samples as in B). Lane 1–5, YPAD; lane 6–10, MM; hours of growth are labeled on the top of each lane. Band intensities (below blots) are given as percentages relative to that of the YPAD/1 day blots. Each lane was developed with 4 (Oxyblot for whole cell lysates), 15 (Oxyblot for mitochondrial extracts) and 16 (Western blot against Sod2p for whole cell lysates) μg of protein.

Figure 8

Model of Fe trafficking in yeast cells. Fe from the medium enters via high-affinity (R_HI) and low-affinity (R_LO) pathways. R_LO is unregulated while R_HI is regulated by the concentration of cytosolic Fe (Fe_C), indicated by the dashed lines. Fe_C moves into the mitochondria and vacuoles, forming mitochondrial (Fe_M) and vacuolar (Fe_V) forms. The import of Fe_C into the mitochondria is regulated by the concentration of Fe_M. In version C, Fe^III nanoparticles (Fe_P) were assumed to form in cytosol whereas in version V, they were assumed to form in vacuoles. Rate-constants and other parameters are listed in Table 1.

Figure 9

Simulated kinetics of Fe uptake and trafficking in yeast cells. A, YPAD, 1-day-growth scale; B, YPAD, 5-day-growth scale; C, MM, 1-day-growth scale; D, MM, 5-day-growth scale. The first 5 hr (before the orange line) reflect exponential growth conditions while the remaining 115 hrs indicate slow-growth and steady-state modes. Black, pink, cyan, yellow, and brown lines indicate [Fe_cell], [Fe_V], [Fe_M], [Fe_C], and [Fe_P], respectively. Dashed line simulates [Fe_cell] when cell growth was set to zero while Fe import rates were set to those for exponential growth.