Proteotoxicity in any cellular compartment leads to a decline in respiration

In order to induce protein stress in several different cellular compartments, we independently deleted a gene copy of three protein chaperones: cytosolic nascent polypeptide associated complex (NAC, EGD2), HSP70 chaperone from the endoplasmic reticulum (erHSP70, LHS1) and mitochondrial HSP70 (mtHSP70, SSC1). Interestingly, regardless of the compartment of protein stress origin, all mutants experience the increase in cytosolic Hsp90 levels, relative to the WT (Fig. 1a, Supplementary Fig. S1).

Figure 1 Chaperone deletion mutants are characterized by increased proteotoxicity and repressed mitochondrial activity. (a) Hsp90 levels are increased in the chaperone deficient mutants relative to the control. **p < 0.01 (ANOVA plus post hoc). (b) Oxygen consumption rates are decreased in the chaperone deficient mutants. Oxygen consumption is measured polarographically in the YPD growth medium supplemented with 2% glucose at 30 °C. (c) Mitochondrial membrane potential-related fluorescence via DiOC6(3) is decreased. After collapsing the ΔΨm by 10 min preincubation with 100 μM CCCP and 5 μg/mL antimycin, signal intensity decreased, indicating mitochondrial membrane depolarization. Mitochondrial biomass is unchanged in the chaperone deficient mutants. The NAO fluorescent signal of energized mitochondria was collected in the appropriate channel following staining. (d) ATP levels are decreased in the chaperone deficient mutants, without change in ROS levels. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). (e) Drop test results display no difference in growth on YPEG medium between the studied strains. Growth on YPEG plates is preceded by growth in the same liquid medium. Left panel displays results of the exponentially growing cells and the right one of the stationary cells. (f) Blue Native gels showing the level of Complexes II, IV and V in the wild type and the chaperone deficient strains. Full size image

At this point, we were interested to characterize a cellular process fundamental for proper cell functioning. Therefore, we set out to determine the impact of proteostasis failure in different compartments on mitochondrial respiration. First, we measured O 2 consumption in yeast mid-log phase in 2%-glucose media, mimicking flask culture conditions. The deficiency of each chaperone led to a significant decrease in O 2 consumption in comparison to the WT strain (Fig. 1b), rendering it similar to the respiration-deficient petite strain (included as a negative control). Furthermore, DiOC6(3) stained chaperone deficient strains displayed a significant decrease in fluorescent intensity, relative to the WT strain (Fig. 1c), indicating a decline of mitochondrial membrane potential (MMP). Since O 2 consumption can also decline as a result of decreased mitochondrial mass, we used a flow cytometry based assay where nonyl acridine orange (NAO) fluorescence is indicative of cardiolipin content, in turn correlating with the mitochondrial mass. No significant difference in NAO fluorescence intensity was observed among the studied strains, indicating that there has been no change in mitochondrial mass (Fig. 1c). This observation is further corroborated by the decreased levels of ATP in the deletion mutants compared to the control, accompanied by unchanged levels of ROS (Fig. 1d).

Next, we tested the possibility that the observed decline in respiration is a result of a chaperone deficiency-related respiratory chain (RC) defect. We therefore evaluated the growth of the wild type and the mutant strains in the YPEG medium (3% ethanol, 3% glycerol), where only respiration competent cells are able to grow, while respiration defective cells are selected against. We observed no significant difference in growth between the wild type and the mutant strains in exponential or stationary phase (Fig. 1e), prompting us to conclude that the observed decrease in respiration is not a result of defective respiratory chain complexes. To further investigate the steady state levels of the mitochondrial respiratory chain complexes, mitochondria were solubilized in the mild detergent Digitonin and subjected to the Blue Native PAGE analysis. We found no significant difference in RC composition between the strains (Fig. 1f).

Localized proteotoxicity leads to a cell-wide response

The observed decline in respiration motivated us to measure trends in gene expression using qPCR in several canonical stress response pathways related to all three relevant compartments: mitochondrial retrograde response, ERAD and cytosolic heat shock response (Fig. 2). The most prominent feature of the chaperone deficient strains is the upregulation of the cytosolic HSP26, normally in charge of targeting proteins to either aggregation or refolding during heat shock (Fig. 2a). Interestingly, the level of HSP42, a small HS protein of similar function, remains unchanged, as well as Hsp70 (SSA1), usually a hallmark of cytosolic HS response activation.

Figure 2 Localized chaperone deficiency does not activate any of the key cellular stress responses. Transcript levels of (a) cytosolic, (b) ER and (c) mitochondrial target genes in the chaperone deficient strains, compared to the WT. UBC6 was used as a control. The measurement was performed in biological and technical triplicate. (d) NADP/NADPH ratio is decreased in the chaperone deficient mutants. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). Full size image

Similarly, in the case of typical ERAD components, only proteins involved in the ER luminal folding displayed an increase in expression (KAR2, PDI1 and SEC62) while protein degradation remained largely unaffected (DER1, HRD1, UBC1) (Fig. 2b). The difference is most prominent in the case of KAR2 (2–4 fold increase in the chaperone deficient strains), while PDI1 and SEC62 display a smaller but significant increase of 1.5–2 fold (Fig. 2b).

Moreover, antioxidant protection was slightly upregulated (1.5–2.5 fold) in both the cytosol (SOD1) and the mitochondria (SOD2) (Fig. 2a,c), indicating leakage of ROS into the cytosol, probably due to changes in the RC activity or the existence of a feed-forward loop preventing the dysfunctional mitochondria from putting the cell under oxidative stress. An imbalance in the expression of the complex IV subunits could cause such a change in the RC activity: while COX4 expression was increased (2-fold in average), the levels of COX1 and COX3 remain unchanged (Fig. 2c).

The cell-wide response includes an increase in the cytosolic NADPH reducing power

Since retrograde response, a cellular response to mitochondrial dysfunction, may have an effect similar to the one observed in the mutant strains, we wanted to rule out its involvement. Considering that the levels of peroxisomal citrate synthase involved in glyoxylate cycle (CIT2) were unchanged (Fig. 2c), we concluded that there is no evidence of retrograde response involvement. However, an interesting metabolic feature is put forward: the upregulation (1.5–2 fold) of the mitochondrial citrate synthase (CIT1) and the downregulation (app. 0.5–0.7 fold) of isocitrate dehydrogenase (IDH1) likely leads to accumulation of citrate. Further, evidence of activation of mtUPR (HSP60) and degradation by mitochondrial Lon protease (PIM1) was absent. However, MCX1, a mitochondrial chaperone with poorly described function was upregulated (Fig. 2c).

In order to characterize the potential consequences of the changes in the TCA cycle enzyme levels, we measured the NADP+/NADPH ratio, whose decrease usually points towards increase in the reducing power in the cytosol. Even though certain variability is present, NADP+/NADPH ratio is significantly decreased in the chaperone deficient strains relative to the control (Fig. 2d).

Taken together, our data suggest that localized proteotoxicity induces a response across the entire cell, one that includes metabolic changes and activation of folding maintenance machinery in multiple compartments. We therefore termed the response cross-organelle stress response (CORE).

Localized proteotoxicity leads to mitochondrial fragmentation

Further, we set to determine the levels of respiratory chain components by Western blot (Fig. 3a, Supplementary Fig. S2). We tested the steady state levels of Qcr8, a subunit of Complex III, the Complex IV components Cox1 and Cox5, the Complex IV assembly factors Coa3 and Cox15, as well as the Complex V subunit Atp5. While Atp5 and Cox15 showed no change in its level upon chaperone deficiency, the integral subunits of Complex III and Complex IV components displayed a different behavior (Fig. 3a, Supplementary Fig. S2). Although only 2-fold in average, Qcr8 and Cox5 levels showed an increase only in the EGD2 and SSC1 deficient strains, while Cox1 and Coa3 level increased in all chaperone deficient mutants. In addition, we tested the steady state levels of the translocase of the outer mitochondrial membrane (TOM) Tom40 and subunits of the translocase of the inner mitochondrial (TIM) membrane Tim21 and Tim23 (Fig. 3a, Supplementary Fig. S2). We found that none of the components displayed any variation between the wild type and the mutants. Furthermore, no clear difference was observed after decoration against the ubiquinol-cytochrome-c reductase protein (Rip1).

Figure 3 CORE pathway is partially a response to respiration decline. (a) Western blot analysis of several respiratory chain components. Porin was used as a loading control. (b) Representative images of cells with visualized mitochondria via preCOX4-mCherry and preSU9-GFP. (c) CORE pathway transcript level in the Coa3-null mutant as well as the wild type treated with 25 μM CCCP. (d) Hsp90 levels are unaffected by the CCCP treatment in the wild type strain. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). (e) Venn diagram of the overlap between the responses in chaperone deficient strains, Coa3-null mutants and the CCCP-treated wild type cells. Full size image

In order to visualize mitochondrial morphology, as well as protein import into mitochondria, we employed MitoLoc plasmid, which allows localization analysis of differentially imported fluorescent marker proteins10. Briefly, GFP is fused to the fungal mitochondrial localization signal of the F 0 -ATPase subunit 9 (preSU9) of Neurospora crassa, whose signal is independent of the mitochondrial membrane potential (MMP), while mCherry is fused to the N-terminal localization sequence of cytochrome C oxidase 4 (COX4) and is imported into mitochondria proportional to the MMP. We found that mitochondria in chaperone deficient strains are smaller and more round than the tubular mitochondria found in WT, which is indicative of mitochondrial fragmentation (Fig. 3b). More specifically, analysis of the mitochondrial volume using MitoLoc ImageJ plugin revealed a distribution shift towards smaller volumes in chaperone deficient mutants relative to the wild type. No significant increase has been detected in the fraction of cells with accumulation of mCherry-Cox4 in the cytosol, suggesting that Cox4 is successfully translocated into the inner mitochondrial membrane (Supplementary Fig. S3). Moreover, both fluorescent signals always co-localized in a similar fraction of cells in both the wild type and the chaperone deficient mutants (Fig. 3, Supplementary Fig. S3). These results suggest that defective protein import into the mitochondria of the chaperone deficient mutants, which could explain the partial upregulation of cytosolic chaperones, is an unlikely scenario.

However, we have considered this possibility further. We thus measured the expression levels of the likely components of the CORE pathway using qPCR in: (i) respiration defective mutant with inactivated COA3 gene, encoding for an assembly factor of the Complex IV and (ii) the wild type strain treated with CCCP, thus abolishing the obligatory linkage between the respiratory chain and the phosphorylation system.

In the case of COA3-null mutant, only HSP26 upregulation was in common with the proposed CORE pathway (Fig. 3c). Furthermore, uncoupling of the respiratory chain with CCCP yielded a response partially overlapping with the response observed in chaperone deficient mutants. It included upregulation of Hsp26 chaperone and the protein disulfide isomerase, PDI1. Additionally, CCCP treatment results in citrate synthase (CIT1) upregulation, albeit without accompanying downregulation of IDH1 as in the chaperone deficient strains (Fig. 3c). However, CCCP treatment yielded other changes, which are not observed in the framework of the proposed CORE pathway: increase in the expression level of the mitochondrial Hsp60, a sharp decrease in the expression level of other ERAD components, SEC62 and KAR2, as well as in SOD1. CIT1 and IDH1 experienced an increase in the expression level, unlike in the context of the proposed CORE pathway (Fig. 3c). In addition, CCCP treatment of the wild type strain did not affect the Hsp90 level (Fig. 3d, Supplementary Fig. S4). These results confirm that the overlap between the CORE pathway and RC uncoupling or an RC defect is minor and that the CORE pathway is unlikely a response to defective protein import and only in small part a response to the decline in respiration.

CORE pathway partially relies on Hsf1 activation

In order to investigate the role of Hsf1 in the regulation of the proposed pathway, we measured the expression levels of the likely constituents in the double deletions of Hsf1 in combination with each studied chaperone using qPCR. We found that in the absence of Hsf1, chaperone deletions did not result in the upregulation of Hsp26 (Fig. 4a) and Hsp90 (Fig. 4b, Supplementary Fig. S5). On the other hand, the oxygen consumption (Supplementary Fig. S5) and the metabolic features of the proposed CORE pathway remained at the level observed in the chaperone deficient strains (Fig. 4a), suggesting that this part of the pathway is regulated in a different manner.

Figure 4 Hsf1 regulates a part of the CORE pathway. (a) Transcript levels of genes proposed to be involved in the CORE pathway. UBC6 was used as a control. The measurement was performed in biological and technical triplicate. (b) Hsp90 levels remain unaffected by the deletion of studied chaperones in the absence of Hsf1. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). Full size image

CORE pathway activation extends replicative and chronological lifespan

Cellular stress responses, when countering mild stress, often have overall beneficial effects on a cell, rather than providing merely a survival route. Therefore, we have set out to measure the replicative lifespan (RLS) of the studied chaperone deficient mutants. RLS is measured as the maximum number of generations that each mother cell goes through before the onset of senescence. The control strain produced a maximum of 19 buds during its RLS, which corresponds to the expected value for this strain (Fig. 5a). The largest effect on RLS with a 40% lifespan extension, in comparison to the control, resulted from the deletions of EGD2, encoding a subunit of the nascent polypeptide associated complex (NAC), as well as SSC1, mtHSP70. Finally, the deletion of LHS1, erHsp70, resulted in 30% lifespan extension relative to the control. Furthermore, we monitored the chronological lifespan (CLS) of the studied strains, measured as the mean and maximum survival time of non-dividing yeast populations (Fig. 5b). As with the replicative lifespan, we found that the chronological lifespan was extended in all chaperone deficient mutants, with the largest effect in the deletion of LHS1 (app 40%), followed by the deletion of EGD2 with 25% extension (Fig. 5b). As in the case of RLS, the smallest effect was observed in the deletion of the SSC1, with only 15% extension (Fig. 5b).

Figure 5 Replicative and chronological lifespan are extended in the chaperone deletion mutants. (a) Replicative lifespan is assessed by micromanipulation. The number of cells is 104, 117, 115 and 121 for the wild type, ∆EGD2, ∆LHS1 and ∆SSC1, respectively. The data shown are pooled from 2 independent experiments for each strain. Significance of the results was tested with log-rank test, p-values < 0.05. (b) Chronological lifespan is assessed by measuring survival in post-mitotic yeast culture every three days by plating. The results represent mean and standard deviation of three independent experiments. Full size image

In summary, both RLS and CLS extensions confirm the beneficial role of the CORE pathway in the context of cellular aging.