Eukaryotic cell biology is temporally coordinated to support the energetic demands of protein homeostasis

Abstract

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H⁺ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.

Fig. 1. Rhythms in oxygen consumption and protein abundance at three dilution rates.

a The period (τ) of oscillation, which is made up of stages of higher and lower O₂ consumption (HOC, LOC), varies with dilution rate (mean ± SEM are used throughout, n = 4 biologically independent samples). Samples were harvested at the times shown. b the duration of LOC, not HOC, varies with dilution rate (extra sum-of-squares F test: straight line vs. horizontal line fit, p_HOC = 0.07, n = 4 biologically independent samples). c Of 3389 proteins detected by quantitative mass spectrometry, only 4% were consistently rhythmic (varied by >33% across all conditions). d Heatmap showing mean abundance of consistently rhythmic proteins clustered with either LOC or HOC (see also Supplementary Data 1). e Oxygen consumption and mean-normalized protein abundance, for representative examples of HOC (high-affinity sulfate permease, Sul1) and LOC (monocarboxylate transporter, Jen1) phase proteins. Source data are provided as a Source Data file.

Fig. 2. Variation of metabolites, transport, and soluble protein across the YRO.

a There are consistent phase relationships between intracellular free amino acids (AA), soluble (Sol) protein, trehalose (storage carbohydrate), AMP, betaine, K⁺, OCR and H⁺ export under all conditions (n = 4 biologically independent samples, two-way ANOVA_Time p value shown, mean ± SEM). b Calibration curve for firefly luciferase emission ratiometric reporter of intracellular (cytosolic) pH. c Intracellular pH (pH_IC) oscillates as a function of YRO phase under all conditions (representative data). d Summary of key events that occur during HOC and LOC. Source data are provided as a Source Data file.

Fig. 3. Switching occurs between protein synthesis (HOC) and autophagy (LOC).

a Model: cells accumulate carbohydrates (CH₂O)_n, amino acids and osmolytes during LOC and consume/export them in HOC to sustain translational bursts and maintain osmostasis. Replete stores increase H⁺ export, a pH-dependent checkpoint activating TORC1 and releasing BMC proteins. HOC ends when stores are exhausted, see Fig. 5e and Supplementary Table 1 for more details. b Puromycin incorporation assay and immunoblot for TORC1 activation (phospho-Rps6, Ser235/236) reveal translational bursting in HOC (n = 3 biologically independent samples, TWA_INT: two-way ANOVA_INTERACTION, total protein loading control). c Immunoblots for cleaved/full-length Pgk1-GFP reveal increased autophagy during LOC (n = 3 biologically independent samples, OWA one-way ANOVA, total protein loading control). d Differential variation in vacuole volume vs. surface area:volume ratio (two-sided unpaired t-test, n = 7 independent experiments for HOC and LOC, n > 68 cells per image). Data throughout presented as mean ± SEM where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. e Acute inhibition of protein synthesis (CHX, 25 µg mL⁻¹ cycloheximide or TORC1 activity 200 nM rapamycin) during HOC immediately terminates HOC and abolishes the YRO, representative OCR and H⁺-export traces are shown. Source data are provided as a Source Data file.

Fig. 4. YROs regulate stress granules and glycogen, and are sensitive to H⁺ and K⁺.

a The intensity and distribution (STD/mean) of stress granule marker Pab1 (Pab1-GFP signal) varies over the YRO, with more foci during LOC and more diffuse during HOC, supporting dynamic variation in stress granule formation (The scale bar represents 1 µm. OWA, n_T90h = 4 images and n_T92-97h = 8 images one experiment, n ≥ 72 granules per time point). b Cellular glycogen stores increase during LOC and decrease during HOC (OWA, n = 3 biological replicates). Liquidation of storage carbohydrates is likely to fuel translational bursting during HOC. c, d Decreasing extracellular pH reduces the period of the YRO duration (representative OCR, n_pH3.4 = 4 or n = 3 independent experiments). e, f HSP30 mutants have truncated oscillations (maroon/red, pH_IC; black/grey, representative OCR, n = 4 biological replicates). g, h Extracellular K⁺ concentration determines YRO period duration (representative OCR). This is unlikely to be due to loss of cell viability as YROs are rapidly restored when potassium becomes available (representative OCR). All data are shown as mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. The YRO regulates resistance to heat stress and protein homoeostasis.

a Viability of cells removed from the bioreactor after heat treatment (55 °C, 2 min) is greatest at the end of LOC, when the abundance of trehalose and osmolytes are greatest. Percentage of heat-treated cells, corrected for viability of non-heat-treated cells harvested at the same time (OWA, n = 3 biological replicates). b Sensitivity of HOC protein synthesis rate to pH and hyperosmotic stress assayed by puromycin incorporation (gly, 10% glycerol; srb, 1 M sorbitol, n = 4 biological replicates). c Strains deficient in glycogen synthesis (gsy2) or glycogen breakdown (gph1) do not initiate YROs and, d, gph1 strains accumulate aggregated protein, showing that glycogen breakdown is necessary for proteostasis. Representative silver-stained gel (two-sided unpaired t-test, n = 4). e A detailed, testable and experimentally derived model for the YRO. Green arrows/lines represent activation/repression, red arrows represent ATP production/stimulation of ATP production, black arrows represent predicted metabolic flux, see key for further details. Data are shown as mean ± SEM. Source data are provided as a Source Data file.