Deletion of sulfate transporter SUL1 extends yeast replicative lifespan via reduced PKA signaling instead of decreased sulfate uptake

Reviewer #1 (Public Review):

The manuscript by Long et al. focused on SUL1, a gene encoding a sulfate transporter with signaling roles in yeast. The authors claim that the deletion of SUL1, rather than SUL2 (encoding a similar transporter), extended yeast replicative lifespan independent of sulfate transport. They also show that SUL1 loss-of-function mutants display decreased PKA activity, indicated by stress-protective carbohydrate accumulation, relevant transcription factor relocalization (measured during aging in single cells), and changes in gene expression. Finally, they show that loss of SUL1 increases autophagy, which is consistent with the longer lifespan of these cells. Overall, this is an interesting paper, but additional work should strengthen several conclusions, especially for the role of sulfate transport. Specific points include the following:

- What prompted the authors to measure the RLS of sul1 mutants? Prior systematic surveys of RLS in the same strain background (which included the same sul1 deletion strain they used) did not report lifespan extension in sul1 cells (PMID: 26456335).

- Cells carrying a mutant Sul1 (E427Q), which was reported to be disrupted in sulfate transport, did not have a longer lifespan (Figure 1), leading them to conclude that "lifespan extension by SUL1 deletion is not caused by decreased sulfate uptake". They would need to measure sulfate uptake in the mutants they test to draw that conclusion firmly.

- Related to my previous point, another simple experiment would be to repeat the assays in Figure 1 with exogenous sulfur added to see if the lifespan extension is suppressed.

- There needs to be more information in the text or the methods about how they did the enrichment analysis in Figure 2B. P-values are typically insufficient, and adjusted FDR values are reported from standard gene ontology platforms (e.g., PANTHER).

- It is somewhat puzzling that relocalization of Msn2 was not seen in very old cells (past the 17th generation), but it was evident in younger cells. The authors could consider another possibility, that it was early and midlife experiences that made those cells live longer. Past that window, loss of Sul1 may have no impact on longevity. A conditional shutoff system to regulate SUL1 expression would be needed to test the above, albeit this is probably beyond the scope of this report.

- The connections between glucose restriction, autophagy, and sul1 (Figure 4) could be further tested by measuring the RLS of sul1 cells in glucose-restricted cells. If RLS is further extended by glucose restriction, then whatever effects they see should be independent of glucose restriction.

- They made and tested the double (sul1, msn2) mutants, but they should also test the sul1, msn4 combination since Msn4 functions similarly to Msn2.

Reviewer #2 (Public Review):

Summary:

In this study, the authors find that the deletion of a sulfate transporter in yeast, Sul1, leads to the extension of replicative lifespan. They investigate mechanisms underlying this extension and claim that the effects on longevity can be separated from sulfate transport, and are instead linked to a previously proposed transceptor function of the Sul1 transporter. Through RNA sequencing analysis, the authors find that Sul1 loss triggers activation of several stress response pathways, and conclude that deletion of two pathways, autophagy or Msn2/4, partially prevents lifespan extension in cells lacking Sul1. Overall, while it is well-appreciated that activation of Msn2/4 or autophagy is beneficial for lifespan extension in yeast, the results of this study would add an important new mechanism by which this could achieved, through perceived sulfate starvation. However, as described below, several of the experiments utilized to support the authors' conclusion are not experimentally sound, and significant additional experimentation is required to support the authors' claims throughout the manuscript.

Strengths:

The major strength of the study is the robust RNA-seq data that identified differentially expressed genes in cells lacking Sul1. This facilitated the authors' focus on two of these pathways, autophagy and the Msn2/4 stress response pathway.

Weaknesses:

Several critical experimental flaws need to be addressed by the authors to more rigorously test their hypothesis.

(1) The lifespan assays throughout the manuscript contain inconsistencies in the mean lifespan of the wild-type strain, BY4741. For example, in Figure 1A, the lifespan of BY4741 is 24.3, and the extended lifespan of the sul1 mutant is 31. However, although all mutants tested in Figure 1B also have lifespans close to 30 cell divisions, the wild-type control is also at 30 divisions in those experiments as well. This is problematic, as it makes it impossible to conclude anything about the lifespan extension of various mutants with inconsistencies in the wild-type lifespan. Additionally, the mutants analyzed in 1B are what the authors use to claim that loss of the transporter does not extend lifespan through sulfate limitation, but instead through a signaling function. Thus, it remains unclear whether loss of sul1 extends lifespan at all, and if it does, whether this is separable from cellular sulfate levels.

(2) While the authors use mutants in Figure 1 that should have differential effects on sulfate levels in cells, the authors need to include experiments to measure sulfate levels in their various mutant cells to draw any conclusions about their data.

1. Similar to point 2, the authors focused their RNA sequencing analysis on the deletion of sul1 and did not include important RNA seq analysis of the specific Sul1 mutation or other mutants in Figure 1B that do not exhibit lifespan extension. The prediction is that they should not see the activation of stress response pathways in these mutants as they do not see lifespan extension, but this needs to be tested.

(4) While the RNA-seq data is robust in Figure 2 as well as the follow-up quantitative PCR and trehalose/glycogen assays in 2A-B, the follow-up imaging assays for Msn2/4 localization in Figure 2 are not robust and are difficult to interpret. The authors need to include more high-resolution imaging or at least a close-up of the cells in Figure 3C.

(5) The autophagy assays utilized in Figure 4 appear to all be done with a C-terminal GFP-tagged Atg8 protein. As C-terminal GFP is removed from Atg8 prior to conjugation to phosphatidylethanolamine, microscopy assays of this reporter cannot be utilized to report on autophagy activity or flux. Instead, the authors need to utilize N-terminally tagged Atg8, which they can monitor for vacuole uptake as an appropriate readout of autophagy levels. As it stands, the authors cannot draw any conclusions about autophagy activity in their studies.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Long et al. demonstrated that the deletion of SUL1, which encodes a sulfate transporter localized on the plasma membrane, extends the replicative lifespan in S. cerevisiae. The authors further investigated the mechanism underlying this lifespan extension. They found that, unlike sul1∆ mutants, other mutants that have been shown to have a deficiency in sulfate transport cannot extend lifespan, from which they concluded that it is unlikely that SUL1 deletion extends lifespan by impairing sulfate intake. The authors then performed a series of characterizations on sul1∆ mutants and found that consistent with previous studies, PKA activity is downregulated when SUL1 is deleted. The authors demonstrated that SUL1 deletion promotes the nuclear localization of Msn2, as well as autophagy, which are known downstream signals of the PKA pathway. In addition, the authors show that MSN2 and ATG8 are indispensable for the lifespan extension in sul1∆ cells. Altogether, this manuscript suggests that SUL1 deletion extends lifespan by affecting PKA activity.

Strengths:

This study reported an interesting phenotype that the deletion of SUL1, but not SUL2, promotes lifespan extension in budding yeast. The authors performed some characterizations on sul1∆ mutants and epistatic studies to demonstrate that this lifespan extension requires MSN2 and ATG8, which further support the importance of the PKA pathway in regulating lifespan.

Weaknesses:

However, one of the major findings in this paper that SUL1 deletion extends lifespan independently of its role in sulfate uptake was merely based on lifespan measurements on sul2∆, SUL1E427Q, and met3∆ mutants, which cannot exclude the possibility that yeast lifespan is affected by sulfate intake. In addition, the strength of evidence for whether SUL1 deletion extends lifespan through affecting PKA activity is incomplete. It has been shown that Sul1 and Sul2 have redundant functions in both sulfate transport and PKA activation (Kankipati et al. 2015). However, in this manuscript, as shown by the authors, the deletion of SUL2 does not extend the lifespan compared with sul1∆ mutants. Without a further characterization on why deletion of SUL1, but not SUL2, extends lifespan, it is likely that SUL1 deletion extends lifespan independently of either sulfate transport or PKA activation.

Author Response

We appreciate your constructive feedback on our manuscript entitled “Deletion of sulfate transporter SUL1 extends yeast replicative lifespan via reduced PKA signaling instead of decreased sulfate uptake” (ID: eLife-RP-RA-2023-94609). Your comments/suggestions are very helpful for improving our manuscript. In particular, we feel additional experiments and analysis suggested by the reviewers will help strengthen our argument that Sul1 deletion mutant extends lifespan via decreased PKA signaling, instead of via decreased sulfate uptake. Below we outline our response to the reviewer's comments/suggestions and the plans for additional experiments and analysis.

(1) Our current model is that lifespan extension following SUL1 knockout depends on the PKA signaling pathway but not sulfate transport. To further substantiate this, we plan to conduct further transcriptome sequencing and dynamic sulfate uptake experiments using WT, Sul1D and Sul1E427Q strains. If our model is correct, we expect that PKA signaling pathway will be more repressed in Sul1D strain than in Sul1E427Q strain, but the sulfate transport will be similar in both strains. This will add strong evidences supporting the model in addition to the lifespan data.

(2) The reviewer mentioned the disparities observed between the lifespan of WT in Figure 1B and other experimental assays. Although it is known that lifespan for WT varies considerably from experiment to experiment (thus the need for WT control for every lifespan measurement), we agree it is important to make a solid conclusion that Sul1E427Q does not extend lifespan. We plan to measure the lifespan of more cells for the mutant strains illustrated in Figure 1B and update the data and charts.

(3) Other issues, for example, the small images of Msn2/4 in the nucleus, grammar and formatting errors, and the lifespan data of double (Sul1/Msn4) mutants will be addressed in the revised version of the manuscript after we performed the additional experiments/analysis.