β-Oxidation and are critical energy providers during acute depletion in Saccharomyces cerevisiae

Carmen A. Webera,1, Karthik Sekarb,1, Jeffrey H. Tanga, Philipp Warmerb, Uwe Sauerb, and Karsten Weisa,2

aDepartment of Biology, Institute of , ETH (Eidgenössische Technische Hochschule) Zurich, 8093 Zurich, Switzerland; and bDepartment of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland

Edited by Jennifer Lippincott-Schwartz, Janelia Farm Research Campus, Ashburn, VA, and approved April 3, 2020 (received for review August 8, 2019) The ability to tolerate and thrive in diverse environments is para- Previous studies have predominantly focused on - mount to all living organisms, and many organisms spend a large induced metabolic changes that occur over hours or longer (8, part of their lifetime in starvation. Upon acute glucose starvation, 12, 13). Yet, metabolic pools rapidly deplete and fill within yeast cells undergo drastic physiological and metabolic changes and seconds (14, 15), and any initial responses have likely been missed reestablish a constant—although lower—level of energy production in these studies. Furthermore, metabolic changes can occur much within minutes. The molecules that are rapidly metabolized to fuel faster than transcriptional and abundance changes (16), energy production under these conditions are unknown. Here, we and metabolism is therefore likely among the first responders to combine metabolomics and genetics to characterize the cells’ re- changes in the environment. However, the rapid metabolic sponse to acute glucose depletion and identify pathways that en- changes that occur at the onset of starvation have remained un- sure survival during starvation. We show that the ability to respire is clear, and the critical energy sources that are utilized upon acute essential for maintaining the energy status and to ensure viability glucose starvation—leading to a new energy equilibrium within ’ during starvation. Measuring the cells immediate metabolic response, minutes (9)—have not been well characterized. Additionally, it is we find that central metabolites drastically deplete and that the in- poorly understood how metabolism communicates with the rest of tracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. the cell to ensure long-term survival.

Furthermore, we detect changes in both and lipid metab- GENETICS To begin to address some of these questions, we performed a olite levels. Consistent with this, both bulk autophagy, a process that metabolomic analysis upon acute glucose starvation in budding frees amino acids, and lipid degradation via β-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, yeast. Our results reveal that upper glycolysis metabolites de- both these pathways ensure long-term survival during starvation. plete within seconds of acute glucose starvation while in- Thus, our results identify bulk autophagy and β-oxidation as impor- tracellular lipid pools increase or are maintained. In addition, we tant energy providers during acute glucose starvation. found that the metabolic flow through amino acid metabolites changes significantly. Based on these findings we assessed the Saccharomyces cerevisiae | acute glucose starvation | metabolomics | effect of lipid digestion and autophagy on cellular energy status lipidomics | β-oxidation Significance ellular life depends on metabolic substrates for growth and Csurvival. Glucose is a common substrate and convenient en- From mammalian cells to microbes, cells need to be able to ergy source that feeds directly into glycolysis, leading to rapid cell quickly respond to changes in their environment, e.g., to growth and proliferation in many microbes. Budding yeast, for changes in nutrient availability. Since metabolic pools can example, depend highly on glucose as an energy source for rapid rapidly deplete and fill within seconds and cellular metabolism growth. One of their evolutionary advantages is that they can is one of the first layers of response to nutrient starvation, we outgrow many of their competitors when supplied with glucose. characterize here the immediate metabolic response after The dependence on glucose and the ability to respond to glucose glucose starvation in yeast cells within seconds as well as starvation not only is common in microbes and unicellular or- hours. Our results reveal the predominant cellular metabolic ganisms, but also pertains to multicellular eukaryotes. A large pathways that are affected by the acute glucose withdrawal, number of cancer cells, for example, rely mostly on glucose and and we demonstrate that bulk autophagy and lipid degradation glycolysis even when oxygen is present (i.e., the Warburg effect), via β-oxidation act in parallel to ensure energy maintenance and which leads to outcompeting of noncancerous cells in terms of are critical for survival under acute glucose starvation conditions. proliferation. Furthermore, starvation from glucose and other carbon sources is especially detrimental for cell types such as Author contributions: C.A.W., K.S., J.H.T., U.S., and K.W. conceptualized and organized the project; C.A.W. designed, performed, and analyzed all survivability assays and ATP neurons, which depend on an uninterrupted carbon supply. In- luminescence-based assays; C.A.W., K.S., and P.W. prepared extracts for mass spectrome- terestingly, energy deficits and low glucose levels have been cor- try measurements; K.S. and P.W. performed mass spectrometry measurements; C.A.W., related with neurodegenerative diseases such as Alzheimer’s K.S., and P.W. analyzed and interpreted the mass spectrometry data; and C.A.W., K.S., (1–3). Thus, understanding how cells cope with starvation is cru- and K.W. wrote the paper. cial for elucidating both normal cellular processes as well as ab- The authors declare no competing interest. errant behaviors in disease. This article is a PNAS Direct Submission. In microbes such as the budding yeast Saccharomyces cer- Published under the PNAS license. evisiae, starvation is especially ubiquitous (4) and must be coun- Data deposition: All ion data as well as custom scripts for the respective figures are pro- teracted very rapidly. Budding yeast have developed an intricate and vided via GitHub (https://github.com/karsekar/yeast-starvation). rapid response to glucose starvation, including reduction of tran- 1C.A.W. and K.S. contributed equally to this work. scriptional and translational activity (5–7), autophagy of cytoplasmic 2To whom correspondence may be addressed. Email: [email protected]. components and lipids for energetic needs (8), and reduction of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ macromolecular diffusivity (9, 10). Starvation states can differ doi:10.1073/pnas.1913370117/-/DCSupplemental. depending on the type of starvation or nutrient limitation (11).

www.pnas.org/cgi/doi/10.1073/pnas.1913370117 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 upon acute glucose starvation and found that lipid degradation candidate substrates during starvation, namely extracellular me- and autophagy act together to ensure ATP maintenance and are tabolites as well as intracellular reserves. important for cellular survival in acute glucose starvation. Extracellular metabolites either could be used as an external energy source or point to the activity of intracellular pathways Results based on secreted metabolites. To examine changes in extra- Respiration Is Crucial for Survival and Energy Maintenance upon cellular metabolites, we grew cells to OD 0.5 to 0.8 in synthetic Acute Glucose Starvation. Our previous results suggested that complete medium with dextrose (SCD), essential amino acids, respiration is critical for energy production upon acute glucose and a yeast nitrogen source (see Materials and Methods for de- withdrawal (9). To address the question of how important res- tails). The cells were then acutely starved by washing in the same piration activity is for the acute glucose starvation response, we synthetic media lacking dextrose (glucose). The supernatant was genetically deleted CBP2 in order to abolish aerobic respiration. sampled before and after the transition, and a control experi- The CBP2 gene product facilitates the splicing of the cytochrome ment was conducted by washing the cells with SCD to ensure B oxidase pre-RNA (17). Therefore, a knockout of CBP2 inhibits that medium change itself did not lead to secondary metabolic the ability of cells to respire. In cbp2Δ mutants, intracellular ATP effects. The strongest effect that we observed was a rapid de- levels dropped to nondetectable levels within 1 h of acute glu- pletion of extracellular amino acids within the first hour of acute cose starvation and remained comparably low for 19 h (one-way glucose starvation—specifically, two exemplary amino acids, as- ANOVA test, 1- and 19-h comparison: P value = 0.12). Addi- partate and methionine (Fig. 2A). Monitoring their abundances tionally, we used Antimycin A, which specifically and rapidly in- beyond this first hour, no additional appreciable changes could hibits the electron transport chain in mitochondria, avoiding any be observed for up to 19 h. Since our glucose starvation medium potential adaptation effects that might have occurred in the long- contains essential amino acids, this could suggest that extracel- term absence of respiration in cbp2Δ cells. Similar to the cbp2Δ lular amino acids are assimilated and catabolized during acute mutant, ATP levels decreased strongly within 1 h of starvation and starvation. To test whether the extracellular amino acids serve as remained low (one-way ANOVA test, 1- and 19-h comparison: P energy sources on a short timescale during glucose starvation, we value = 0.8). Furthermore, the survival rate during starvation in measured intracellular ATP levels within the uptake period of 1 h or within longer starvation up to 19 h in cells starved of the absence of respiration was dramatically reduced (Fig. 1A). − This showed that respiratory activity is critical to provide the glucose (SC), and starved of glucose and amino acids (SC AA). necessary energy, which correlated with survival during starvation. Furthermore, we starved cells by washing them in water (H2O) to assess the effect of any extracellular component (i.e., all nitrogen Metabolite Pools Deplete Globally within 1 h of Starvation in Cells sources) present in the SC medium (synthetic complete medium Unable to Respire. Having established that respiration is required without glucose) (Fig. 2C). The ATP levels did not drop much for energy maintenance and survival upon glucose starvation (Fig. lower in water nor in the medium lacking glucose and amino 1A), we next aimed to identify the substrate(s) feeding respiration acids compared to medium lacking only glucose, for neither under these conditions. We hypothesized that respiration sub- rapid (30 min) nor short (19 h) starvation duration. strates cannot be efficiently metabolized in a respiration-deficient To complement the ATP measurements with functional evi- mutant and hence might accumulate in these cells. We therefore dence, we also conducted spotting assays for cells starved in SC media, in SC media without amino acids (SC–AA), and in water measured and compared central carbon metabolites between wild- (H O) to assess survival and recovery after the starvation stress. type and cbp2Δ cells on a timescale of 1 to 4 h of starvation (Fig. 2 Cells starved from both glucose and amino acids initially showed 1B). A large swath of intracellular metabolites exhibited similar no survival difference compared to cells starved from glucose depletion patterns between the two strains, including many central only (up to day 7 of starvation) and even seemed to have a metabolites in glycolysis (e.g., -phosphate, - survival advantage after 14 d of starvation (Fig. 2B and SI Ap- bisphosphate, and glucose-6-phosphate) and in the citric acid cy- pendix, Fig. S1). Survival of cells washed in water instead of SC cle (e.g., acetyl-CoA, glutamate, glutamine). Some select metab- or SC−AA was only slightly and insignificantly impaired after 14 olites, such as glucose-6-phosphate, acetyl-CoA, ribose-5-phosphate, Δ to 21 d compared to the glucose-starved cells (Fig. 2B and SI and glutamate, depleted more completely in the cbp2 mutants Appendix, Fig. S1). Although extracellular amino acids were compared to wild type, and others, including sedoheptulose-7- taken up within 1 h of starvation, we thus conclude that they guanine, phosphoenolpyruvate, and UDP-hexose, diminished en- neither affected ATP levels upon acute glucose starvation nor Δ tirely during glucose starvation in cbp2 but remained constant or provided cells with a survival advantage long term. even accumulated in wild-type cells. However, we could not detect Another potential candidate substrate during glucose starva- Δ any intracellular metabolite that specifically accumulated in cbp2 tion is intracellular glycogen. Under various starvation condi- cells upon glucose starvation. We also noted the energy charges tions, intracellular glycogen resources were proposed to build up (ATP, ADP, and AMP) and observed varying patterns between or to be used for energy maintenance (18). Since glycogen was the two strains; specifically, in wild type, the abundances equili- shown to decrease to nonmeasurable levels after glucose starva- brated within 2 h (e.g., ADP and AMP) or depleted less severely tion (19), we tested whether intracellular glycogen resources sus- (ATP). In cbp2Δ, the measured charges fluctuated wildly (e.g., tain ATP maintenance and survival during short-term starvation. ATP) or were more depleted compared to wild type (e.g., ADP, However, a mutant deficient in glycogen synthesis (glg1Δglg2Δ)did AMP), suggesting a dysregulation of for re- not adversely affect ATP levels after 1 to 19 h of acute glucose spiratory mutants. We conclude that intracellular metabolite pools starvation and showed no significant survival deficiency even after deplete rapidly in both wild-type and respiration-deficient cells 21 d (Fig. 2 D and E and SI Appendix,Fig.S5). Thus, glycogen and do so even more rapidly or more completely when cells are stores in the cell are not a main energy source upon acute glucose unable to respire. starvation.

Maintenance of ATP Levels and Increased Survival under Acute Central Carbon Metabolites Exhibit Subminute Changes to Glucose Glucose Starvation Are Explained neither by Extracellular Amino Deprivation. Our initial survey did not provide us with good Acids nor by Intracellular Glycogen. Our initial analyses of intracellu- candidate substrates that yeast cells utilize to ensure their sur- lar metabolites did not provide us with obvious candidates fuel- vival upon acute glucose starvation, but suggests that many major ing respiration that became apparent through accumulation in a metabolic changes occur on a timescale faster than 1 h. Hence, respiration-deficient mutant. We next focused our attention on our experiments thus far might have missed initial responses that

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913370117 Weber et al. Downloaded by guest on September 26, 2021 ATP ATP AB100 WT cbp2Δ 1 h WT +AntA ADP ADP

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G6P G6P Glutamine Glutamine ard) Guanine Guanine and tandard s internal standar internal internal internal internal st internal

ed ed GMP GMP l Glutamate Glutamate PEP PEP labe e f th o nt of the labeled u

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Ion count ofthe label UDP-hexose UDP-hexose ( unt/ ount/(Ion co ount/(Ion o c Ion Ion c Ion count/(Ion count

Acetyl-CoA Acetyl-CoA R5P R5P Time (hours) Time (hours) WT cbp2Δ non-starved Time (hours) Time (hours) Time (hours) Time (hours) Glucose starved

Fig. 1. The ability to respire is crucial for survival and energy maintenance upon acute glucose starvation. (A) Relative ATP levels in the respiratory-deficient cbp2Δ mutant and wild-type cells treated with the respiration inhibitor Antimycin A, compared to wild type after 1 and 19 h of acute glucose starvation. Mean, SD, and biological replicates are shown. (Right) Survival after 1 h, 3 d, and 6 d of acute starvation for wild-type (WT), cbp2Δ mutants, and wild-type cells treated with Antimycin A (WT + AntA) during starvation. (B) Relative change in energy charges during starvation. Change in ion intensity for ATP, ADP, and AMP after 0, 1, 2, 3, and 4 h of acute glucose starvation (red, glucose starved), compared to nonstarved cells (blue, nonstarved). Comparison between WTand cbp2Δ cells. Average and SE (error bar) of two biological replicates are shown. (C) Change in ion intensity for metabolites after 0, 1, 2, 3, and 4 h of acute glucose starvation (red, glucose starved), compared to nonstarved cells (blue, nonstarved). Comparison between WT cells and cbp2Δ cells. FBP, fructose bisphosphate; G6P, glucose-6-phosphate; Glycerol P, Glycerol phosphate; GMP, guanosine monophosphate; Ms1P, mannose 1-phosphate; R5P, ribose-5- phosphate; S7P, sedoheptulose-7-guanine; PEP, phosphoenolpyruvate. Average and SE (error bar) of two biological replicates are shown.

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Fig. 2. Extracellular amino acids and intracellular glycogen levels are not the main short-term energy source upon acute glucose starvation. (A) Extracellular aspartate and methionine deplete primarily within the first 1 h of starvation. Wild-type and cbp2Δ mutants were first grown in SCD (synthetic complete medium with glucose) and then switched to SCD or SC (synthetic complete medium without glucose), indicated by time point 0 h. Extracellular samples were taken and measured for relative abundance of ions corresponding to aspartate and methionine. Error bars indicate the SE of two biological replicates.(B)

Survival of wild-type yeast cells after 18 h to 21 d of acute glucose and amino acid starvation (SC−AA) and acute starvation in water (H2O), compared to survival of cells only starved from glucose (SC). (C) ATP levels after hours and minutes of starvation in SC, SC–AA, or H2O. (D) ATP levels of glg1Δglg2Δ mutants compared to wild-type after 19 h of acute glucose starvation. (E) Quantified spotting assays after 2 h to 21 d of starvation. Mean and SD and values of biological replicates are shown.

could be important for long-term survival. Therefore, we ex- down in Antimycin A-treated cells, leading to less generation of panded our metabolomic analyses to earlier time points. Since amino acids. These observations echoed our earlier observations metabolic pools can deplete and fill within seconds (14, 15), we about respiratory-deficient cells with generally lower metabolic aimed for measurements on a 10- to 60-s timescale. We employed pools over long timescales. a high-throughput, untargeted mass spectrometry approach (15, Glycolysis metabolites such as hexose phosphates decreased 20) to measure the global, starvation-induced metabolic changes under both conditions within 10 s. Intermediates of the pentose that occur in budding yeast. Yeast cells were cultivated in SCD phosphate pathway such as ribose phosphates (corresponding ∼ media, and using a rapid filtration setup (21, 22), we dispensed 1 to the pentose phosphate ion) and potential precursors such as OD unit of yeast on a filter, perfused with SCD media for at least D-ribose (pentose) also decreased under both conditions, but 10 s followed by exposure to media without glucose (SC) for varying times (10, 20, 30, and 60 s) (Fig. 3A). The yeast-containing were then maintained at lower levels in the untreated cells while filters were rapidly quenched after the designated exposure time, they depleted even more under the respiratory-deficient condition and intracellular metabolites were extracted. Relative changes of (Fig. 3C), correlating with the decreased phosphoenolpyruvate intracellular metabolite concentrations were measured and an- levels observed in the cbp2Δ cells at later time points (Fig. 1B). notated (20). This semiquantitative method allowed us to measure To further substantiate these observations in wild-type untreated the response of ∼350 ions that can be associated with 650 me- cells, we obtained metabolite concentrations with a targeted mass- tabolites known to the metabolome of Saccharomyces cerevisiae spectrometry method (25) and found other glycolytic and pentose (23). Rapid fold changes occurred in many metabolites visualized phosphate pathway metabolites to exhibit a similar rapid depletion on a metabolic map (Fig. 3B). (Fig. 3D), specifically dihydroxyacetone phosphate, glyceraldehyde How does respiration affect the metabolism at these early time 3-phosphate, and fructose bisphosphate. The rapid depletion was points? To answer this question, we treated cells with Antimycin comparable to an earlier study, examining Escherichia coli carbon A and measured them in parallel with untreated cells in our fast starvation (14). These results suggest that, upon removal of the filtration setup. An increase of NADH in Antimycin A-treated glucose input downstream, metabolic activity continues, leading cells confirmed the successful application of the drug, since the to the successive depletion of metabolic pools. Such depletion is block of the electron transport chain inhibits NADH oxidation expected to start with upper glycolysis metabolites as the entry (24) (Fig. 3C). Glucose, as indicated by the hexose ion, de- creased within the first 10 s under both starvation conditions, point for glucose. indicating that the cells use up internal glucose very rapidly upon The energy charges of the cells (e.g., ATP, ADP, and AMP) starvation. showed similar rapid changes (Fig. 3E). ATP depleted within Furthermore, the Antimycin A treatment in starvation led to a seconds, and AMP increased in a near-equivalent time frame. Our stronger decrease of most amino acid pools compared to cells data suggest that global metabolic changes occur within seconds of that were only starved (Fig. 3C and SI Appendix, Fig. S2), in- starvation. dicating that these metabolite pools are either degraded and Interestingly, we observed that both intracellular amino acid potentially explored for energy generation much faster than in and lipid pools changed considerably within these initial time points. cells capable of respiration or that protein degradation slows We therefore focused on these two classes for our further studies.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913370117 Weber et al. Downloaded by guest on September 26, 2021 Central Nucleotide A B carbon log2 Lipids change 2.4 2 mL of OD ~0.5 Growing yeast AminoAmin 0 acidacid Perfuse on filter Quench, extract, and Pre Treatment measure on -4.5 0s FIA QTOF 10s 20s 30s -10s 60s Exposure times C SCD SC SC + Antimycin A NADH Hexose Hexose-Phosphate Pentose Pentose-Phosphate 3 0 0 0 0 0 **

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Exposure time (seconds) Exposure time (seconds) GENETICS Fig. 3. Central carbon metabolites exhibit subminute changes to glucose deprivation. (A) Fast filtration setup. Growing yeast (∼1 OD*mL) were deposited onto filters and quickly perfused with pretreatment media (SCD). At the 0-s time point, cells were perfused with either SCD or SC (starvation) medium, and after exposure for a given amount of time (10, 20, 30, and 60 s), the filters were rapidly quenched in extraction solution and measured on mass spectrometry for small metabolites (m/z < 1,000 g/mol). (B) A metabolic map of central carbon metabolism shows rapid depletion of metabolites in upper glycolysis and increase in lipid synthesis. The log2 change in ion intensity is shown between 60 s exposure of SC media versus the average of the 10- and 30-s exposure on SCD media. (C) Glycolytic metabolites deplete strongly within 60 s upon exposure to SC media. The log2-adjusted values of intensity for specific ions are shown and labeled according to the annotated compound. Measurement time indicates the exposure time for the given media for the cells on the filter (SCD, blue; SC, red; SC with Antimycin A, gray). Six dots are shown for each time point (three biological replicates and two technical measurement replicates). Time point 0 s is extrapolated from the average of the SCD condition. Error bars indicate the SE for three biological replicates. Significances are shown between the SC and SC with Antimycin A condition for time 60 s (* 0.01 < P < 0.05; **10−4 < P < 0.01, and ***P < 10−4) and calculated via Student’s t test. (D) Measurement of other metabolites (DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; FBP, fructose bisphosphate) with another mass spectrometer quantification method also corroborate the depletion of upper glycolysis metabolites. Two dots are shown for each time point (two biological replicates). Error bars indicate the SE for two biological replicates. (E) ATP, ADP, and AMP levels as indicators of cellular energy homeostasis rapidly change during starvation.

Autophagy Is Important for Energy Maintenance upon Acute Glucose the vacuole reveals that autophagy is activated within 19 h of Starvation and Ensures Long-Term Survival. Since our map of glucose starvation (SI Appendix, Fig. S4). We then generated a starvation-induced metabolite changes revealed changes of amino mutant incapable of organizing the preautophagosomal structure acid metabolites, we examined whether amino acid digestion atg2Δ (27). Since bulk autophagy and the cytoplasm-to-vacuole could provide a pathway for ATP production through respiration transport (CvT) pathway rely largely on the same machinery, and whether intracellular , which are synthesized prior to including ATG2, the atg2Δ mutant will affect additionally any starvation, could be an amino acid source upon acute glucose specific process that relies on the CvT for shuttling to the vac- removal. Since proteasomal degradation is one of the main spe- uole and degradation therein. Importantly, the deletion of ATG2 cific protein degradation pathways, we explored whether protea- did not affect the cells’ ability to respire and grow on non- somal degradation contributed to ATP generation and maintenance fermentable carbon sources such as glycerol (SI Appendix,Fig.S3) within 1 h of glucose starvation. To test this, proteasomal activity but led to impaired autophagy levels both in glucose starvation and was blocked using the small molecular inhibitor MG132. ATP nitrogen starvation (SI Appendix,Fig.S4). ATP levels after 19 h of concentrations in MG132-treated cells compared with the control glucose starvation significantly decreased in the atg2Δ mutant cells showed no significant changes upon glucose starvation for 1 h compared to wild-type cells (Fig. 4B). In addition, fewer atg2Δ cells (Fig. 4A). Due to its potential cellular toxicity, we did not test the survived 7 d of glucose starvation in comparison to the isogenic effect of MG132 on long-term survival, but our results neverthe- wild-type control (Fig. 4C and SI Appendix,Fig.S5). Thus, bulk less suggest that proteasomal degradation is not a significant en- autophagy and specific autophagic processes involving the CvT ergy source during the first hours of acute starvation. pathway contributed to energy maintenance, correlating with sur- Another pathway that frees building blocks and carbon sub- vival in acute glucose starvation. strates is bulk autophagy, which removes cytosolic components, Our data further implied that there must be alternative path- nonspecifically triggering their degradation in the vacuole. We ways to generate ATP in the short term as autophagy deficiency therefore examined whether bulk autophagy is important for the did not decrease ATP levels to the same levels as seen in the cellular response to glucose starvation. First, we used GFP-Atg8 complete absence of respiration (Fig. 4B) and did also not fully as a marker for autophagy (26). The appearance of free GFP in impair long-term survival.

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Fig. 4. Unlike proteasomal activity, bulk autophagy contributes to survival and energy maintenance upon acute glucose starvation. (A) ATP levels after 1 h of pretreatment with MG132 or DMSO as a control, followed by 1 h in acute SC or SCD medium with MG132/DMSO only. (B) ATP levels in cbp2Δ and atg2Δ relative to WT cells after 1 and 19 h of acute glucose starvation. (C) Survival of atg2Δ cells after 2 h to 14 d of glucose starvation. Means, SDs, and biological replicate values are shown. Significances are shown between the wild-type and mutants (ns, indicates nonsignificant; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P > 0.0001; and ****P < 0.0001). Significance was calculated using a one-way ANOVA test followed by a Holm-Sidak test.

β-Oxidation Is Required for Energy Maintenance and Survival upon mutants deviated only slightly from values measured in wild-type Acute Glucose Starvation Together with Autophagy. In addition to cells (Fig. 6A). However, consistent with the results obtained by amino acids, lipid metabolite levels changed on these very rapid Seo et al. (8), acute complete glucose starvation led to survival timescales, and we observed a rapid increase in some lipids in deficiency after 7 d (Fig. 6B). We conclude that μ-lipophagy wild-type untreated cells, such as hexadecanoic acid and mediated by Atg14 did not contribute significantly to energy 3-oxooctanoyl-CoA (Fig. 5A). There are two potential non- maintenance within the first 19 h of acute glucose starvation, but exclusive models that could explain the changes that we see. On had long-term effects on survival of the cells after 7 d. one hand, since a majority of lipids in the cell originates from While μ-lipophagy specifically may contribute only to energy membranes, the observed changes could be caused by global maintenance after the first day of starvation, general lipid di- membrane remodeling during starvation. On the other hand, the gestion in peroxisomes could play a role early on. β-Oxidation cells might liberate lipids to use them as an energy/carbon source occurs exclusively in peroxisomes in yeast (31). To attenuate during starvation, leading to an increase of free fatty acyls. To test the first model, we utilized a lipid extraction method global lipid degradation in peroxisomes, we deleted the gene designed for yeast (28) and measured the lipidome over time coding for Pot1. Pot1 is the only 3-ketoacyl-CoA thiolase in yeast β (Fig. 5 B and C), sampling cells before glucose withdrawal and that catalyzes the last step of -oxidation, producing acetyl-CoA Δ every 10 min after. We could annotate over 10,000 ions by exact to feed into the (32). Indeed, pot1 cells showed mass matching to over 400 classes in the eight categories within decreased ATP levels within 19 h of glucose starvation, dem- the LIPID MAPS scheme (29) and classified the different classes onstrating that β-oxidation of fatty acids contributes to ATP as increasing or decreasing (Fig. 5B). In our dataset, the three maintenance upon glucose starvation (Fig. 6A). Furthermore, main membrane lipid classes (sphingolipids, phospholipids, and survival of pot1Δ cells decreased after 7 to 14 d in the absence of sterol lipids) were enriched consistent with the fact that the glucose (Fig. 6 B and C and SI Appendix, Fig. S5). majority of lipids in cells originates from membranes (30). No Together, our data suggest that lipid consumption by major differences appeared between the lipids measured under β-oxidation contributes to intracellular ATP levels upon acute control versus glucose starvation conditions. Nonetheless, a few glucose starvation within several hours of stress and correlates traces were found to change, and particularly, lipids annotated to with a benefit for long-term survival. By contrast, μ-lipophagy, as dolichols and linear polyketides exhibited an upward trend a more specific way to consume lipids, does not seem to con- during starvation (Fig. 5D). While this suggests that specific tribute to short-term ATP maintenance, but is important for lipids and lipid classes may be synthesized or depleted in direct long-term survival. response to starvation, the overall lipid composition and there- Similar to the results for the autophagy-deficient mutant, the fore cellular membranes did not seem to appreciably change ATP and survival levels were not completely abolished in the within rapid timescales (30 min or less). This argues that yeast pot1Δ mutant and did not reach the levels of the respiratory- cells do not undergo a major membrane remodeling during acute deficient cbp2Δ mutant. We therefore wondered whether bulk glucose starvation. autophagy and β-oxidation might complement each other and Since our results did not support the hypothesis that the generated a pot1Δatg2Δ double-deletion strain. Consistent with changes that we saw in lipid metabolites were caused by a major the hypothesis that the two pathways contribute in an additive reorganization of cellular membranes, we hypothesized and ex- amined whether the liberated lipids were used as an energy manner to energy generation upon short-term glucose starvation, source to fuel respiration during starvation. μ-Lipophagy was we observed that after 19 h of starvation the double-deletion recently shown to play a role during long-term glucose restriction strain showed lower levels of intracellular ATP than each of from 2% (wt/vol) to 0.4% (wt/vol) glucose (8). The study sug- the single-deletion mutants (Fig. 6 A and C). While we observed gests that lipid droplets are digested through microlipophagy in some experiments also a survival deficiency in the double mediated by Atg14 to ensure survival in limiting glucose condi- compared to the single mutants, this difference was not statisti- tions. We therefore tested whether μ-lipophagy is also necessary cally significant. We conclude that lipid degradation and auto- for ATP level maintenance upon acute complete glucose star- phagy work in parallel to ensure energy maintenance upon vation. Within 19 h of starvation, the ATP levels in atg14Δ glucose starvation.

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Exposure time (s) CD GENETICS

Fig. 5. The lipidome during rapid starvation. (A) Hexadecanoid acid and 3-oxooctanoyl-CoA as examples of lipid-related metabolites that accumulate within 10 to 60 s. The log2-adjusted values of intensity for specific ions are shown and labeled according to the annotated compound. Measurement time indicates the exposure time for the given media for the cells on the filter (SCD, blue; SC, red). Six dots are shown for each time point (three biological replicates and two technical measurement replicates). Time point 0 s is extrapolated from the average of the SCD condition. Error bars indicate the SE for three biological replicates. (B) Experimental setup for measuring yeast lipidomics upon starvation entry. Exponentially growing yeast cells (OD 0.8 in SCD media) were sampled (time point 0 min) and then resuspended in fresh SCD or SC media. Samples were taken every 10 min thereafter. All samples were processed (see Methods) and measured. Putative lipids were annotated based on m/z and correspondence to the LIPID MAPS database. (C) The normalized distribution of annotated lipids using the LIPID MAPS identifiers. The eight major lipid categories are shown. Error bars indicate the SE between two biological replicates. (D) The lipid classes linear polyketides (PK01) and dolichols (PR0307) were identified as accumulating in glucose starvation compared to nutrient-rich conditions. Error bars indicate the SE between two biological replicates.

Discussion important for survival of these cells. Our data agree with the latter, Cells commonly experience sudden changes in nutrient availabil- show that autophagy is induced within the first 19 h of starvation, ity; therefore, strategies to overcome nutrient scarcity are critical and additionally suggest that energy levels, as measured by ATP, to ensure survival in periods of starvation. However, the imme- decrease in autophagy-deficient cells already within 19 h. diate metabolic response to starvation remains poorly character- In the respiratory-deficient cbp2Δ mutant, we found sedo- ized. Here, we aimed to identify the main immediate energy heptulose-7-guanine (S7P), guanine, and phosphoenolpyruvate resources of budding yeast upon acute glucose starvation. We (PEP) to deplete during starvation in direct contrast to wild-type showed that respiration is needed for survival and energy main- cells. S7P, guanine, and PEP are all potentially connected through tenance upon glucose starvation, and we examined the metabolic , the pentose phosphate pathway, and nucleotide resources that are fed into the respiratory chain. We found that, synthesis. Low PEP concentration would suggest that the cbp2Δ within seconds of glucose starvation, upper glycolysis metabolites mutants have lower gluconeogenic flux (35), which also correlates decrease drastically. Furthermore, we show that autophagy and with the decreased ribose levels observed for rapid timescales (10 β-oxidation are critical energy providers as early as within the to 60 s) in the Antimycin A-treated starved cells. High concen- first day and play a central role for survival of yeast during long- tration of PEP is needed in order to drive the pathway to glucose- term starvation. 6-phosphate and consequently through the pentose phosphate The role of autophagy in starvation for glucose-grown cells is pathway (where S7P resides), eventually becoming nucleotides controversial. A few studies conclude that autophagy is neither (e.g., guanine). Gluconeogenic synthesis of PEP is primarily cat- activated nor necessary for short-term survival during starvation alyzed by PEP carboxykinase. In yeast this reaction is driven by (12, 33), whereas other studies suggest the opposite (8, 34). We GTP hydrolysis. Given the lower levels of energy cofactors and observed that autophagy is essential for cell survival after glucose presumably of energy fluxes in cbp2Δ cells (Fig. 1A), the pro- starvation within 7 d and that ATP levels depend on autophagy duction of PEP by carboxykinase is likely down-regulated (36). within the first 24 h of starvation. While Adachi et al. (33) sug- An earlier study concluded that μ-lipophagy is triggered by gest that autophagy is not induced within a few hours of glucose glucose deprivation via the global energy regulator AMPK (8) within starvation, they also show that within days autophagy becomes several days of starvation. Yeast AMPK activity is known to directly

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100 100

50 50 % ATP compared to WT 0 % ATP compared to WT 0

∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ WT WT pot1 atg2 cbp2 pot1 atg2 pot1 atg14∆ cbp2 ∆pot1∆ atg14 ∆ atg2 atg2 B atg14∆ pot1∆ C ns ** * ns *** **** **** **** *** * ns

WT 100

100 o pared t m

50 50 ival co v %sur %survivalcomparedtoWT 0 0 2 h 7 d 14 d 2 h 7 d 14 d ∆ ∆ ∆ ∆ WT t1 po atg2 cbp2 ∆pot1∆atg14 ∆glg2∆ atg2 glg1

Fig. 6. Lipid degradation and autophagy ensure survival and energy maintenance upon acute glucose starvation. (A) ATP levels in mutants compared to WT after 1 or 19 h of acute glucose starvation. (B)Survivalofatg14Δ and pot1Δ cells after 2 h to 14 d of acute glucose starvation. (C) Survival of mutant cells compared to WT cells after 14 d of acute glucose starvation. Significances are shown between the wild-type and mutants (ns, nonsignificant; *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P > 0.0001; and ****P < 0.0001). Significance was calculated using a one-way ANOVA test followed by a Holm- Sidak test.

correlate with increased AMP/ATP ratio (37). We had observed UDP-hexose (presumably UDP-glucose) in cbp2Δ cells within 1 changes in the energy charges of the cells (e.g., ATP, ADP, and to 4 h (Fig. 1B). In cbp2Δ cells, all potential intracellular energy AMP) (Fig. 3E) congruent with the observations of Seo et al. (8). sources, including UDP-hexoses, might be drained within hours Strikingly, we measured ATP to deplete within seconds while AMP of starvation due to the lack of efficient energy generation via increased in a near-equivalent time frame. This activity may suggest respiration, while wild-type cells might refrain from degrading that AMPK potentially activates earlier and conveys the intracellular metabolites needed for rapid regrowth after starvation. The in- signaling to manage starvation within seconds. While AMPK sig- crease of dolichol in wild-type cells within 30 min of starvation, naling has been long studied, the kinetics of induction have not been on the other hand, could point to an inhibition of glycosylation entirely revealed (38). Our data open up the possibility that AMPK upon carbon starvation. Indeed, previous evidence suggests that signaling may be activated within seconds of starvation. the dolichol pathway is transcriptionally down-regulated in re- One of the expected responses from AMPK signaling is an sponse to glucose starvation (41, 42). increase in β-oxidation activity. While a direct inhibition of Atg14 In this paper, we sought to better characterize the strategies dependent μ-lipophagy did not affect the general energy status that yeast cells employ as they enter starvation. Our results show within the first 19 h of starvation, we found that the ability to β that yeast starvation is multifaceted and entails responses at many perform general -oxidation is important for ATP maintenance as β well as survival in acute glucose starvation and that lipid inter- levels (e.g., metabolites, energy level) and pathways ( -oxidation, mediates increase within seconds after glucose starvation. Our autophagy). We conclude that multiple metabolic responses are lipidomics dataset suggests that these immediate changes are not needed to ensure a sufficient supply of substrates for respiration as due to large-scale membrane remodeling since we find that only a the cells maximize their survivability. few lipid traces increase upon glucose starvation, in particular Methods polyketides and dolichols (Fig. 5D). Polyketides are a structurally very heterogeneous class of compounds of which many have been Strains and Growth. All strains used in this study have a W303 background, and precise genotypes are listed in the SI Appendix, Table S1. Yeast strains attributed to antimicrobial function (39). The cells might manu- were grown in liquid SCD medium (synthetic complete with 2% [wt/vol] facture the polyketides as an antimicrobial measure during star- glucose, containing 6.7 g/L yeast nitrogen base [Difco] and amino acids vation. Less microbial competition for remaining nutrients as well according to SI Appendix,TableS2)atpH5.0(titratedwithHCl/KOH)at30°C as nutrient freeing by eliminating potential competitors could be a rotating. YPD (Yeast extract–Peptone–Dextrose) plates (2% glucose [wt/vol, rational strategy to ensure survival during starvation. Sigma], 2% Bacto peptone [wt/vol, BD], 1% yeast extract [wt/vol, BD], 0.01% The other lipid that increased during 30 min of starvation was Adenine Hemisulfate [wt/vol, Sigma], and 2% Bacto agar [wt/vol, Chemie dolichol, which is required for protein glycosylation in the en- Brunschwig]) or YPGly plates (same as YPD plates, but with 2% glycerol [wt/ doplasmic reticulum (Fig. 5D). Here dolichol functions together vol, Sigma] substituting the glucose and without 0.01% Adenine Hemisulfate) with UDP-glucose as a carrier to deliver substrates for glyco- were used for spotting assays. Gene deletions were performed by homologous sylation (40). Interestingly, we also observed the depletion of recombination of a PCR-amplified cassette encoding antibiotic resistance,

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1913370117 Weber et al. Downloaded by guest on September 26, 2021 functional amino acid-encoding genes, or functional nucleotide-encoding with cells was incubated at −20 °C overnight, and the extraction mix was genes according to SI Appendix, Table S1 (43). subsequently transferred to 15 mL tubes. The extraction solvent was evaporated at 0.12 mbar to complete dryness in a SpeedVac setup (Martin Acute Starvation and Drug Treatments. For acute glucose starvation, cells were Christ), and the samples were dissolved in 100 μL of water and transferred washed three times in SC medium by repeating the following steps three to a 96-well plate for measurement. Samples were stored at −20 °C until times: 1) centrifuging for 1 min to pellet the cells, 2) removing supernatant, and measurement. 3) resuspending the cells in SC medium. Control cells in SCD were treated in the

same way by washing three times in SCD medium. For SC–AA or H2O treatment, Metabolomics Measurement and Annotation. For untargeted analysis (Figs. 2A, cells were washed three times either in SC medium lacking the added amino 3 A–C and E, and 5A), extracts were measured using flow-injection analysis

acids (SC−AA) (SI Appendix,TableS2) or in sterile water (H2O),bothwithapH quadrupole time-of-flight in negative ionization mode and annotated to of 5.0 as in the SCD and SC medium. For nitrogen starvation, cells were washed metabolites as described in a previous study (20). For targeted analysis (Figs. three times in a 1.7 g/L yeast nitrogen base supplemented with 2% glucose. 1B and 3D), measurement and analysis is described in previous work (25). For For MG132 treatment, strains with a deletion of PDR5 were used. Cells were all targeted analysis measurements, an internal standard of fully labeled C13 treated with 100 μM MG132 (Sigma Fluka Aldrich) for 60 min at 30 °C. For extract was used to normalize all data (44). combinations of MG132 treatment and glucose starvation, cells were pre-

treated as described with MG132 and then washed three times in SC medium Lipidomics Extraction. For each sampling, ∼25 OD units of cells (OD600*mL) containing MG132. For Antimycin A treatment, cells were treated with 10 μM were captured on filter paper using a fast filtration setup. The cells with filter Antimycin A (Sigma Fluka Aldrich) in starvation medium. Antimycin A was paper were immediately quenched in 10 mL of yeast lipid extraction solution added to the medium used for washing of the samples as well. (15:15:5:1:0.018% of ethanol, water, diethyl ether, pyridine, and 4.2 N am- monium hydroxide, respectively) at −20 °C. The extraction solution with cells ATP Measurements. ATP measurements were done according to ref. 8 with was incubated at −20 °C overnight, and the extraction mix was subsequently minor modifications. Cells were pelleted and resuspended in 750 μL 90% transferred to 15 mL tubes. The extraction solvent was evaporated using a acetone. Normalization was done according to OD. The cells were then in- SpeedVac setup (Martin Christ), and the samples were dissolved in 100 μLof cubated at 90 °C for ∼10 min to evaporate the acetone, when about 50 μLof 45:45:10% of isopropanol, methanol, and water, respectively, and transferred to solution remained. A total of 450 μL of buffer (10 mM Tris pH 8.0, 1 mM glass vials with inserts. Samples were stored at −20 °C until measurement. ethylenediaminetetraacetic acid) was added to the solution, and ATP was measured with an ATP Determination Kit (Thermo Scientific) on a CLAR- Lipidomics Measurement. Chromatographic separation and analysis by mass IOstar microplate reader (BMG). spectrometry was done using a 1200 series HPLC system with a Phenomenex Kinetex column (1.7 μL × 100 mm × 2.1 mm) with a SecurityGuard Ultra (Part Survival Assays. Cells were acutely starved and incubated at 30 °C rotating. No: AJ-9000) coupled to an Agilent Technologies 6550 Accurate-Mass Q-Tof. Cells were normalized by the OD measured within the first hour of starva- Solvent A: H2O, 10 mM formic acid; Solvent B: acetonitrile, 10 mM formic tion and spotted onto YPD plates, starting at an OD of 0.2, and in a 6 to 8× acid. Ten microliters of extract were injected, and the column (C18) was GENETICS dilution series, diluting 5× at every step. The spotting plates were incubated eluted at 1.125 mL/min. Initial conditions were 60% solvent B: 0 to 2 min; at 30 °C for 1 to 2 d. Percentages of survival compared to wild type were 95% B: 2 to 4 min; and 60% B: 4 to 5 min under initial conditions. Spectra assessed by counting colony forming units (CFUs) per dilution after the were collected in negative ionization mode from 50 to 3,200 mz with high indicated days of starvation, compared with the CFUs counted after the resolution at 4 GHz. Continuous infusion of calibrants (Agilent compounds same number of days in starvation of the wild type. HP-321, HP-921, HP-1821) ensured exact masses over the whole mass range. We converted the raw data files to the mzML format using msConvert and Autophagy Levels during Glucose Starvation. Wild-type and Δatg2 cells processed them in R using the XCMS (ver. 3.0.2) and CAMERA (ver. 1.34.0). expressing GFP-Atg8 were starved from glucose or nitrogen for 19 h, and M-H and M+FA-H ions were annotated using LIPID MAPS (vers. March 2017) thereafter the cells were imaged using an inverted Ti microscope (Nikon) to with a mass tolerance of 0.005 amu (29). check for localization of GFP signal to the vacuole of cells. Data Availability Statement. All data discussed in the paper along with custom Fast Filtration Wash, Sampling, and Extraction. For each measurement, ∼1OD scripts used to generate figures can be found in SI Appendix or on GitHub at unit of cells (OD 600*mL) was captured on filter paper using a fast filtra- https://github.com/karsekar/yeast-starvation. tion setup (21, 22). Immediately, the cells were suffused to flowing pre- treatment media (SCD media) for at least 10 s. Upon media switch, the ACKNOWLEDGMENTS. We thank the U.S. and K.W. laboratory members for feedback and advice; Elisa Dultz, Stephanie Heinrich, and Ruchika Sachdev flow of pretreatment media was stopped, and the posttreatment media for critical reading of the manuscript; John Peter Arun Thomas for advice was followed for a given amount of time (SC media for 10, 20, 30, and 60 s on the lipid extraction; Marieke F. Buffing and Brendan Ryback for fast or SCD for 0 and 20 s). After the posttreatment media, the cells and filter filtration help; and Marieke F. Buffing and Tobias Fuhrer for targeted mass paper were immediately quenched in 4 mL of extraction solution of spectrometry help. This work was supported by Swiss National Science 40:40:20% acetonitrile:methanol:water at −20 °C. The extraction solution Foundation Grant SNF 31003A_179275 (to K.W.).

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