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F U L L T I T L E : Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae.
F U L L T E X T S O U R C E : bioRxiv
ABSTRACT
The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wildtype yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.
INTRODUCTION
Many studies of biological aging are performed with only a few age-points. While these types of studies have revealed a multitude of biological processes that become dysfunctional with age, they are unable to reveal the sequence, kinetics, and penetrance of age-associated changes. Defining these parameters is necessary in order to better understand the network of failures that results in age-associated pathological physiology. In the budding yeast (Saccharomyces cerevisiae), most aging studies have also been performed on populations of cells, lacking the resolution to identify differences within individual cells. Recent developments in microfluidic device technology have allowed for the characterization of yeast replicative aging with whole-lifespan breadth and single-cell resolution (Chen et al., 2017; Crane and Kaeberlein, 2018; Chen et al., 2019). This can allow for the identification of early drivers of aging as well as an understanding of the kinetics and penetrance of these changes, and information about their correlations with ultimate lifespan at single-cell granularity. This information may yield richer insight needed for the development of early interventions to combat age-associated diseases.
The lysosome/vacuole is a central node of cellular metabolism, with critical roles in nutrient sensing and storage, metal homeostasis, organelle maintenance, and protein degradation (Li and Kane, 2009). Accordingly, lysosomal dysfunction underlies many clinically significant genetic and degenerative diseases (Carmona-Gutierrez et al., 2016; Perera and Zoncu, 2016). The lysosome/vacuole is maintained at an acidic pH, which is necessary for proper organelle function. Recent work has shown that the loss of lysosomal/vacuolar acidity is an evolutionarily conserved early life driver of aging and mitochondrial dysfunction (Baxi et al., 2017; Ghavidel et al., 2018; Hughes and Gottschling, 2012), and interventions that increase vacuolar acidity have been found to extend lifespan in yeast (Sasikumar et al., 2019).
The acidic environment of the lysosomal/vacuolar lumen is generated by a highly conserved multi-subunit proton pump, the vacuolar ATPase (V-ATPase). In mammals, genetic ablation of the V-ATPase function is lethal (Inoue et al., 1999). However, in the budding yeast Saccharomyces cerevisiae, deletion of vacuolar ATPase subunits or dedicated assembly factors (vma mutants) is tolerated but results in a variety of physiological dysfunctions. In addition to alkalization of the vacuole, vma mutants exhibit respiratory incompetence (Eide et al., 1993); substantially reduced replicative lifespan (McCormick et al., 2015; Schleit et al., 2013); as well as sensitivity to elevated calcium levels (Ohya et al., 1986), alkaline pH (Serrano et al., 2004), iron depletion (Davis-Kaplan et al., 2004), reactive oxygen species (Milgrom et al., 2007), and DNA damage (Liao et al., 2007). How loss of V-ATPase activity and the resulting alterations in cellular pH homeostasis cause these pleiotropic phenotypes and limit lifespan remains unclear.
In both yeast and metazoans, lysosomal activity is necessary for the maintenance of iron homeostasis (Kidane et al., 2006; Milgrom et al., 2007). Iron is essential for many cellular functions and exists in various forms in the cell, including diiron, heme, and iron-sulfur clusters (ISCs). In yeast, the iron regulon is a coordinated gene expression program that is activated by iron deficiency and results in increased iron uptake and redistributed iron usage (Philpott and Smith, 2013). Interestingly, this program is triggered not by low total iron levels in the cell, but by reduced iron sulfur cluster (ISC) availability (Chen et al., 2004). ISCs are ancient protein prosthetic groups used in a wealth of essential cellular processes including DNA replication and repair, amino acid production, protein translation, and the electron transport chain (Lill et al., 2012). ISC production is dependent on a series of mitochondrial iron-transfer steps (Lill et al., 2012), and it has been speculated that they are the ultimate reason for the persistence of mitochondria in anaerobic eukaryotes. Indeed, mutations in proteins that utilize or assemble iron sulfur clusters underlie a plethora of devastating genetic cancer syndromes, hematopoietic abnormalities, and neurological diseases (Lill et al., 2012). Additionally, during conditions of ISC deficiency, genome maintenance is impaired (Díaz de la Loza et al., 2011; Pijuan et al., 2015; Veatch et al., 2009), mimicking a critical evolutionarily conserved hallmark of aging (López-Otín et al., 2013).
We hypothesized that determining how vacuolar acidity results in the pleiotropic defects and respiratory incompetence of vma mutants would provide insight into how age-associated loss of vacuolar acidity impacts cellular function during aging. Our results indicate that disruption of iron homeostasis is an important hub tying perturbed pH homeostasis and mitochondrial function. Stemming from this observation, we find that wildtype yeast display ISC defects during aging. Interestingly, we observe a divergence in iron-related homeostatic trajectories that cells can undergo during aging. A large subpopulation of cells appears unable to respond to the ISC-related crisis, while other cells mount the expected compensatory gene expression program that allows these cells to survive genomic instability and achieve a full lifespan potential.
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RESULTS
Iron rescues pleiotropic phenotypes following disrupted V-ATPase function
Previous studies suggested that the loss of vacuolar acidity limits lifespan through mitochondrial dysfunction (Hughes and Gottschling, 2012). In order to identify links between vacuolar acidity and mitochondrial function, we performed a multi-copy screen for suppressors of the mitochondrial respiratory deficiency phenotype in vma mutants. We found that overexpression of the FET4 gene encoding for a low-affinity iron transporter allowed vma21Δ cells to grow under respiratory conditions (Figure 1a). An additional screen for spontaneous and UV-mediated suppression of the vma21Δ respiratory deficiency yielded numerous loss-of-function alleles in the ROX1 gene (Figure 1b and Table S2). Another genetic screen for suppressors of vma21Δ sensitivity to an iron chelator also yielded loss of function ROX1 alleles (Table S3). Rox1p is a transcriptional repressor of genes involved in the yeast hypoxic response, so loss of ROX1 results in activation of yeast hypoxic responsive genes. FET4 is one of the most upregulated targets in rox1Δ mutants (Jensen and Culotta, 2002; Ter Linde and Steensma, 2002; Waters and Eide, 2002), and FET4 was required for suppression of vma21Δ respiratory deficiency by mutation of ROX1 (Figure 1c).
Figure 1. Enhanced iron uptake or additional iron allows respiratory growth following impairment of the V-ATPase.
(A) Overexpression of FET4 from a high copy (2µ) plasmid rescues growth of vma21Δ mutants under respiratory conditions. (B) A vma21Δ mutant strain containing a suppressing loss of function mutation in the ROX1 gene (rox1-A10T) rescues the vma21Δ respiratory deficiency phenotype. © FET4 is required for rox1Δ rescue of vma21Δ respiratory growth. (D) Supplemental iron (500 µM ferrous ammonium sulfate) rescues pleiotropic phenotypes of vma21Δ mutants. (E) Chemical inhibition of the V-ATPase with 3 µM bafilomycin A1 preferentially impairs growth under respiratory conditions and iron rescues this phenotype. (F) Replicative lifespan under fermentative growth conditions (YPD media) of wild type or vma21Δ mutants with or without 20 mM sodium ascorbate and/or 0.5 mM iron II sulfate. Where indicated, fermentative growth is on YPD media containing 2% glucose, and non-fermentative respiratory growth conditions is growth on YPG media containing 3% glycerol. Lifespan statistics are shown in Table S7.
Since Fet4 is an iron importer, we also tested supplementation of the growth media with iron, which also resulted in suppression of the vma21Δ respiratory deficiency (Figure 1d). This iron-mediated rescue of vma respiratory growth occurred with different forms of iron (II or III) and in multiple vma mutants lacking either cytosolic (V1) or membrane associated (V0) subunits of the V-ATPase (Figure S1a). Interestingly, iron supplementation additionally rescued other pleiotropic phenotypes of vma21Δ mutants including sensitivity to: elevated pH and calcium, manganese, and oxidative stress induced by paraquat (Figure 1d). Iron supplementation also rescued respiratory defects in wildtype yeast when the V-ATPase is chemically inhibited by bafilomycin (Figure 1e), suggesting that both acute and chronic inhibition of the V-ATPase impairs mitochondrial function by altering iron homeostasis. The short replicative lifespan of vma21Δ and vma13Δ mutants was additionally rescued by addition of iron and/or sodium ascorbate (Figure 1f and Figure S1b). Sodium ascorbate is an antioxidant that can reduce iron from the ferric (Fe3+) to ferrous (Fe2+) form (de Silva et al., 1997). The addition of sodium ascorbate diminished the iron regulon activation in vma21Δ mutants (Figure S1c), rescued the sensitivity of vma21Δ mutants to an iron chelator, and appeared to qualitatively increase the growth rate of wildtype yeast in the presence of an iron chelator (Figure S1d). This ability of sodium ascorbate to impact iron homeostasis may occur in three ways. Conceivably, ascorbate may have beneficial effects by reducing iron in the media (making it more soluble and bioavailable), by reducing iron intracellularly, and/or by acting directly as an intracellular antioxidant.
Reduced V-ATPase function results in iron dyshomeostasis
In yeast, the response to low intracellular iron levels is mediated by nuclear localization of the transcription factor Aft1, which activates the iron regulon, transcription of a suite of genes involved in iron assimilation (e.g., FET3, FTR1, and FIT2). Using an Aft1 responsive transcriptional reporter expressing GFP under the control of the FIT2 promoter (Diab and Kane, 2013), vma21Δ mutants displayed increased levels of GFP fluorescence compared to wild type, which was reduced by the addition of iron (Figure 2a). There is also nuclear localization of Aft1-GFP in vma21Δ cells, and exogenous iron reduced the nuclear localization of GFP tagged Aft1 in vma21Δ cells (Figure 2b). Although vma21Δ cells clearly induce the iron starvation response, we failed to detect any significant change in total intracellular iron levels (Figure 2c), which is consistent with prior reports for other vma mutants (Diab and Kane, 2013; Szczypka et al., 1997).
Figure 2. Iron dyshomeostasis occurs following disruption of the V-ATPase.
(A) Flow cytometry analysis of GFP levels under control of the Aft1 responsive FIT2 promoter of wild type and vma21Δ mutants grown under fermentative conditions (YPD media) with or without 1 mM iron. Values are the mean of 3 samples, each consisting of 20,000 cells per condition. Statistics are shown in Table S8. Error bars represent the standard deviation of the 3 sample means. (B) Fluorescent microscopy images of wild type or vma21Δ mutants in the presence or absence of 100 µg/ml Bathophenanthrolinedisulfonic acid (BPS, iron chelator) or 1 mM iron ammonium sulfate. © Iron levels were measured in wildtype and vma21Δ cells grown in YPD media. No statistical difference was found using Student’s t-test. n=3, error bars represent standard deviation. (D) Aconitase activity was measured for wildtype cells, vma21Δ, and aco1Δ mutants. Each group is statistically significantly different from each other group by ANOVA and Bonferroni’s multiple comparison test P<0.001. n=3, error bars represent standard deviation.
Nuclear localization of Aft1 and activation of the iron regulon are triggered by deficits in iron sulfur cluster availability (Rutherford et al., 2005), suggesting the possibility that ISCs become limiting in vma cells. Aconitase is a mitochondrial ISC containing enzyme in the TCA cycle, and aconitase activity was reduced in vma21Δ cells (Figure 2d). consistent with a prior observation in vma2 mutants (Diab and Kane, 2013).
Taken together, these observations suggested the possibility that disruption of iron homeostasis, and specifically, a deficiency in ISC availability, may fundamentally underlie some of the pleiotropic defects of vma mutant cells. Moreover, these observations suggest that since aging is accompanied by loss of vacuolar acidity, alterations in iron homeostasis and ISC status may occur during aging.
Aging is characterized by a heterogeneous loss of vacuolar acidity coupled to an incompletely penetrant compensatory iron homeostatic response
To characterize the loss of vacuolar acidity during replicative aging, we tagged the vacuolar-localized carboxypeptidase Prc1 (Huh et al., 2003) with a ratiometric pH-sensitive fluorescent protein pHluorin2 (Mahon, 2011). Imaging these cells in a microfluidic device with live-cell fluorescence imaging indicated that loss of vacuolar acidity begins essentially at the start of life (Figure 3a). By comparing different genetic strains, previous studies have indicated that loss of vacuolar acidity limits lifespan (Ghavidel et al., 2018; Hughes and Gottschling, 2012). To test whether vacuolar acidity was a risk factor for early death in a wildtype isogenic population, we monitored changes in vacuolar acidity during early life (ages 0-12 divisions). In concordance with previous observations, the cells which experienced a faster rate of vacuolar acidity loss also exhibited a shorter lifespan (Figure 3b, Table S4), indicating that loss of vacuolar acidity is a predictor of replicative lifespan for individual cells within an isogenic population.
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