August 25, 2022
Perturbations in iron homeostasis affect aging, but how this happens has remained a bit of a black box. A new study by Patnaik et al. in Cell Reports illuminates this box by looking more closely at the transcription factors that are first to respond when iron becomes limiting.
Key among these are Atf1p and Atf2p, which activate the full suite of iron-mobilization genes, among which is TIS11/CTH2, which encodes an RNA-binding protein that targets specific messages for decay.
The targeted messages flagged for decay encode mitochondrial proteins, as these use iron but are not the most essential in the set. The most essential Fe-requiring enzymes are those involved in DNA synthesis and repair, such that slowing/shutting mitochondrial function is a response to iron deficiency. Intriguingly, mitochondrial function also happens to decline with age.
To find the specific mechanisms linking iron with aging, the authors used an unbiased analysis of genes involved in iron homeostasis to see which showed connection with aging. The strain with a tis11Δ mutation lived longer than any others, with a lifespan extended by 51.1%. In a broader sense, they found that genes involved in different aspects of response to iron deficiency also had different effects on fitness and aging.
Delving more deeply into the role of Tis11p/Cth2p in aging, the authors used RNA-seq and Ribo-seq to look at temporal changes in transcription versus translation in aging cells. They showed how, overall, aging leads to inhibition of translation—except for certain genes which are upregulated instead. Interestingly, most of the upregulated genes are in the Fe regulon that gets activated by the first responder Atf1p.
While the expression of TIS11/CTH2 increases both with aging and with iron deficiency, the deletion of the gene extends lifespan. Thus, multiple lines of evidence suggest Tis11p/Cth2p is a negative regulator of longevity. The key connection appears to be mitochondrial translation, where the function of Tis11p/Cth2p to inhibit translation of mitochondrial transcripts for repressing non-essential Fe-requiring enzymes serves to simultaneously repress overall mitochondrial respiration, which speeds aging.
As not all genes translationally upregulated in the tis11Δ mutant contained appropriate binding sites in the 3’ UTR, the authors looked further and found binding sequences for Puf3p, a protein known to bind and inhibit translation of mRNAs coding for mitochondrial ribosome proteins. Thus, Puf3p appears to be a critical partner for Tis11p/Cth2p in mediating downregulation of mitochondrial function. Further, they questioned the relationship with the Hap4p transcription factor, which regulates numerous components of the electron transport chain and whose overexpression extends lifespan. As the combination of a tis11Δ deletion with HAP4 overexpression had no additive effect in an epistasis experiment, they concluded that Tis11p-dependent repression acts through Hap4p.
The role of phosphorylation of Tis11p/Cth2p was examined by mutating N-terminal serine residues, which impairs degradation of the protein. Consistent with the converse result of extended lifespan in null mutants, the nondegradable version of the protein shortens lifespan.
Thus, the ease of the yeast model once more illuminates intricate connections between critical proteins, facilitating potential drug discovery around several new aging factors.
Categories: Research Spotlight
Tags: aging, cell aging, iron homeostasis, Saccharomyces cerevisiae, yeast model for aging
April 22, 2022
Replicative lifespan (RLS), determined by the number of daughter cells a mother cell produces before death, has proved to be an effective model for studying aging in budding yeast. The chromatin-associated proteins Sir2p and Fob1p have been shown to modulate ribosomal DNA (rDNA) and impact the formation of extrachromosomal rDNA circles (ERCs), an accumulation of which is linked to a shorter lifespan.
A recent study by Hotz M et al. in PNAS has shown that chromosomal rDNA copy number (CN) positively correlates with RLS in budding yeast. The authors performed whole-genome sequencing (WGS) of 13 wild-type strains and analyzed the lifespan data, which showed an increased rDNA CN along with enhanced RLS. Additionally, the data showed that the rDNA CN explains the majority (~ 70%) of RLS variation observed in almost identical wild-type strains.
To understand this correlation, the authors analyzed ERC levels in aging cells and fob1-Δ strains, in which ERC levels are low. Together, the analysis concluded that ERCs are inversely correlated with rDNA CN. Exploring further, the authors found that cells with lower rDNA CN showed improved accessibility of the upstream activating factor (UAF) complex binding site at the SIR2 locus. This change in chromatin accessibility reduces the expression of SIR2, causing higher ERC levels and thus a shortened lifespan, implicating both Sir2p levels and ERCs as the underlying cause of the CN-RLS correlation.
Additionally, the authors analyzed the CN-RLS relationship in a set of mutant strains (such as hda2-Δ, upb8-Δ, gpa2-Δ, etc.), all known to increase lifespan. The data showed that while some mutants appeared to impact the CN-RLS relationship, rDNA CN strongly influenced the RLS of these mutant strains (except for fob1-Δ).
Thus, the study demonstrates how rDNA CN impacts yeast lifespan by regulating certain aging factors and highlights rDNA copy number as an essential parameter to examine in aging studies.
Categories: Research Spotlight
Tags: replicative life span, Saccharomyces cerevisiae, yeast model for aging
April 08, 2022
Continuing our theme of highlighting lifespan in yeast, this week’s study by Liu et al. in eLife looks at a possible mechanism for age-related loss of mitochondrial quality. As loss of mitochondrial quality is linked to the rate of building new mitochondria, and as mitochondrial biogenesis depends on the import of new building blocks through the mitochondrial membranes, the abundance of transporters is an important determinant. The authors wondered whether cells were “smart” enough to coregulate the level of transporters with the level of new building blocks.
To get at this question, they overexpressed the translocases of the outer membrane (TOM) proteins to ask if the overabundance of a particular one caused the overabundance of representative mitochondrial proteins, i.e. displayed a regulatory role. Of these, only TOM70 overexpression (OE) increased the abundance of the four representative mitochondrial proteins. They further showed that TOM70 OE led to increased levels of mitochondrial DNA, likely via Mip1p, the mitochondrial DNA polymerase gamma. As further evidence of the role of TOM70 in regulation, the tom70∆ knockout led to the reverse effect from OE, i.e. reduction in levels of both mitochondrial proteins and mtDNA. Tom70p is a well-conserved receptor within the TOM complex, but this role in regulation of mitochondrial biogenesis is a novel finding.
The authors then examined a number of possible intermediate signaling possibilities between Tom70p and the increased abundance of their target proteins. By disrupting various transcription factors and assessing reactive oxygen species, they saw only partial blockage by loss of any one signaling partner, and thus concluded that Tom70p works via multiple pathways.
As TOM70 appears to have a role in regulating new mitochondria, and mitochondrial defects are well established as an indicator of age, the authors asked about connections between TOM70 and aging. They first noted that Tom70p levels go down over time across many organisms. Is this a cause or an association? To counteract the wild-type reductions in Tom70p levels, they overexpressed TOM70 and then examined age-related phenotypes. Higher levels of Tom70p reversed age-associated loss of mitochondrial membrane potential, age-related reductions in other mitochondrial proteins, and, indeed, extended the lifespan of yeast. Further, the tom70∆ knockout once more showed the reverse effects, leading to accelerated aging and reduced lifespan.
Using the remarkable power of yeast, the authors were then able to study the mechanics of the reduction in Tom70p over time. They found that mRNA levels reduce over time due to loss of transcriptional activity, which was rescuable by changes made to the TOM70 promoter. Further, they showed the degradation of Tom70p increased over time due to increased levels of the protein Dnm1p, involved in sorting proteins for degradation under age-related vacuole deacidification. Thus, TOM70 mRNA levels decrease and Tom70p protein becomes less stable, providing redundancy in reduction of a protein that activates mitochondrial biogenesis. With the relative ease possible in yeast, the authors made important headway in revealing these mechanistic links between mitochondrial quality and aging.
Categories: Research Spotlight
Tags: mitochondrial biogenesis, mitochondrial import, mitochondrial quality, mitochondrial regulation, Saccharomyces cerevisiae, yeast model for aging
April 01, 2022
In last week’s post, we discussed asymmetry in pH between mother and daughter cells. This week we’re continuing the theme of mother/daughter inheritance patterns with a study of asymmetrical mitochondrial inheritance. The underlying hope is to garner clues about aging and lifespan, since mother cells have reduced lifespan relative to their daughters.
Yeast cells are normally symmetrical, i.e. round. One of the first steps during cell growth is the breaking of symmetry to create poles, where one pole will become the bud tip and the other pole will become the mother cell tip. In a recent paper in iScience, Yang et al. describe how the mother cell tip is normally distal to the bud tip and that higher-functioning mitochondria localize to both poles. The mitochondrial F box protein Mfb1p was shown to be key for tethering mitochondria at the mother cell tip, which prevents every higher-functioning mitochondria from going to the bud. Mfb1p remains associated with the mother cell tip throughout the entire cycle, and is the only protein known to do this.
Interestingly, Mfb1p is itself asymmetrically localized, where it associates with mitochondria in the mother cell but remains excluded from the daughter cell until just before cytokinesis.
To assess the link between aging and polarity, the authors labeled bud scars with dyes that distinguished between new and old scars. In highly polarized cells, new scars will form directly next to one another. They found polarized bud site selection in >97% of young cells, which was quickly reduced to 89% after 6–10 divisions and plateaued at ~70% for the oldest cells. Thus, aging as a factor of polarity was detectable early in lifespan and continued to decline to a fixed level. Likewise, the polarized localization of Mfb1p to mother cell tips also declined with age, where young cells showed Mfb1p tethered to mitochondria almost exclusively at the mother cell tips, but over time the localization dispersed throughout the mother cell. Interestingly, deletion of RSR1/BUD1 (required for polarized bud site selection) disrupted this polarized localization of Mfb1p within the mother, yet didn’t cause Mfb1p to go to the bud (i.e. cause loss of asymmetry). Thus, polarization and asymmetry could be separated in the bud1Δ mutant cells—and these cells also showed reduced lifespan. The same was seen in bud2Δ and bud5Δ cells.
Given that both bud1Δ and mfb1Δ cells had reduced lifespan, the authors asked whether the two mutations affected lifespan in the same way by examining the double mutant for additive effects. Seeing no additive effects, they concluded that BUD1 and MFB1 operate in the same pathway for controlling mitochondrial distribution and the associated effects on aging.
Whereas the study we highlighted last week did not find a link between aging and pH in yeast, Yang et al. show that mitochondrial localization and inheritance patterns have a clear relationship with replicative lifespan. The secrets of aging continue to invite study, and future results should be equally intriguing.
Categories: Research Spotlight
Tags: mitochondria, mitochondrial inheritance, polarity in yeast, Saccharomyces cerevisiae, yeast inheritance, yeast model for aging
March 25, 2022
Yeast researchers have long observed asymmetry in cytosolic pH between mother and daughter cells. Likewise, researchers have detailed the accumulation of long-lived asymmetrically retained proteins (LARPs) in mother cells, one of which is the plasma membrane proton pump Pma1p. Pma1p transports protons out of the cytosol, thereby increasing pH, and mother cells show marked increases in vacuolar pH as they age. As mother cells have a shorter replicative life span (RLS) than daughter cells, and aging factors have been linked to pH, it is reasonable to ask whether accumulation of Pma1p itself reduces life span in mother cells.
To address this specific question, Yoon et al. in a recent issue of International Journal of Molecular Sciences described screening for suppressors of asymmetric inheritance of Pma1p. They identified three vacuolar protein sorting genes (VPS8, VPS9, and VPS21) for which mutation resulted in high percentages of abnormally symmetric distribution of Pma1p-GFP. As all three of these genes are involved in endocytosis, they asked whether these mutations affected all proteins that are asymmetrically distributed between mother and daughter cells, or just those that reside in the plasma membrane. They found the latter, that the defect is restricted to PM proteins.
Using these mutants with abnormal asymmetric distribution of the proton pump, the authors asked if defects in asymmetric distribution of Pma1p caused changes in aging. Did mother cells without accumulated Pma1p have a longer life span? The answer was a clear “No.” There was little difference in RLS between mutant and wild type, showing that asymmetric distribution of Pma1p does not correlate with aging.
These results are in fact a bit surprising, because aging in mother cells had been previously correlated with a mutation in PMA1 (the pma1-105 allele, Henderson et al., 2014), which increased lifespan by about 30%. Thus, it appears that Pma1p plays a role in aging, but not via asymmetric distribution between mother and daughter cells.
This study adds to what is known about a complicated system. As usual, the awesome power of yeast genetics (#APOYG) provides an excellent forum in which to ask complicated questions in a simple system. Hopefully the links between pH, aging and asymmetry will be revealed in future experiments.
Categories: Research Spotlight
Tags: asymmetry in cell division, replicative life span, Saccharomyces cerevisiae, yeast model for aging