Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation
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Evaluation Summary:
This work provides valuable new information to those who study enzyme mechanisms, nucleotide metabolism, and the response of cells to stress such as nutrient deprivation. The study focuses on CTP Synthase (CTPS), an important enzyme in nucleotide biosynthesis that has been shown to assemble into foci and filaments in yeast cells undergoing starvation conditions. The authors study the structure of yeast CTPS and its propensity to polymerize in low pH (mimicking starvation conditions), and how CTPS filamentation relates to the cellular assemblies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.
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Author Response:
Reviewer #1 (Public Review):
Rarely do I read a paper and have so little to criticize. The paper is very well written and the studies have been conducted carefully and described fully. The only comment that I would make is that the divergence of quaternary structure in CTPS filaments brings to mind some well-known parallels that should be cited and discussed. Perhaps the most prominent one is hemoglobin, where this protein can form tetramers in some species that are very different from the vertebrate tetramers. In plants, I believe that dimeric hemoglobins have been described. Another example would be the diversity of filaments formed by actin-like proteins in bacteria, while in eukaryotes the actin filament architecture has been extremely conserved.
We thank the reviewer for pointing out the evolutionary parallels …
Author Response:
Reviewer #1 (Public Review):
Rarely do I read a paper and have so little to criticize. The paper is very well written and the studies have been conducted carefully and described fully. The only comment that I would make is that the divergence of quaternary structure in CTPS filaments brings to mind some well-known parallels that should be cited and discussed. Perhaps the most prominent one is hemoglobin, where this protein can form tetramers in some species that are very different from the vertebrate tetramers. In plants, I believe that dimeric hemoglobins have been described. Another example would be the diversity of filaments formed by actin-like proteins in bacteria, while in eukaryotes the actin filament architecture has been extremely conserved.
We thank the reviewer for pointing out the evolutionary parallels with well-known polymers, and have updated our discussion with a comparison to diverse actin architecture.
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Evaluation Summary:
This work provides valuable new information to those who study enzyme mechanisms, nucleotide metabolism, and the response of cells to stress such as nutrient deprivation. The study focuses on CTP Synthase (CTPS), an important enzyme in nucleotide biosynthesis that has been shown to assemble into foci and filaments in yeast cells undergoing starvation conditions. The authors study the structure of yeast CTPS and its propensity to polymerize in low pH (mimicking starvation conditions), and how CTPS filamentation relates to the cellular assemblies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Rarely do I read a paper and have so little to criticize. The paper is very well written and the studies have been conducted carefully and described fully. The only comment that I would make is that the divergence of quaternary structure in CTPS filaments brings to mind some well-known parallels that should be cited and discussed. Perhaps the most prominent one is hemoglobin, where this protein can form tetramers in some species that are very different from the vertebrate tetramers. In plants, I believe that dimeric hemoglobins have been described. Another example would be the diversity of filaments formed by actin-like proteins in bacteria, while in eukaryotes the actin filament architecture has been extremely conserved.
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Reviewer #2 (Public Review):
In this work, the authors investigate the role of enzyme polymerization in enzymatic regulation and cellular metabolism. The authors use yeast CTP Synthase (CTPS) in their studies, an enzyme critical for nucleotide biosynthesis and which is also known to form large clusters (self-assemblies) in cells under nutrient deprivation (starvation) conditions. Homologs of yeast CTPS from human and bacteria are known to form linear polymers composed of multiple copies of the CTPS enzyme. Such enzyme polymerization is now known to occur in many other enzyme systems as well, though few in-depth studies such as the current work have been performed. Hence many questions remain including the effect of enzyme polymerization on enzyme activity and regulation, its role in cell biology and metabolism, and its connection to the …
Reviewer #2 (Public Review):
In this work, the authors investigate the role of enzyme polymerization in enzymatic regulation and cellular metabolism. The authors use yeast CTP Synthase (CTPS) in their studies, an enzyme critical for nucleotide biosynthesis and which is also known to form large clusters (self-assemblies) in cells under nutrient deprivation (starvation) conditions. Homologs of yeast CTPS from human and bacteria are known to form linear polymers composed of multiple copies of the CTPS enzyme. Such enzyme polymerization is now known to occur in many other enzyme systems as well, though few in-depth studies such as the current work have been performed. Hence many questions remain including the effect of enzyme polymerization on enzyme activity and regulation, its role in cell biology and metabolism, and its connection to the enzyme self-assembly clusters seen in cells undergoing particular stress responses. The authors use a powerful combination of cryo-electron microscopy, biochemistry, mutagenesis, and yeast studies to investigate these questions for yeast CTPS. The authors succeed in showing that yeast CTPS polymerizes in a pH-sensitive way (low pH mimics starvation conditions), due to a particular histidine/aspartic acid interaction at the interface between CTPS tetramers in the polymer, and that polymerization stabilizes the inactive conformation of the enzyme. Hence, filamentation inhibits yeast CTPS enzyme activity, which is consistent with what would be expected for a biosynthetic enzyme when the cell is under starvation conditions. The authors also succeed in showing the importance of enzyme polymerization to cellular metabolism, as mutations that disrupt or enhance CTPS polymerization slow the growth rate of yeast cells. Interestingly, the authors speculate that polymerization may serve a function beyond merely inhibiting the enzyme under nutrient starvation, but also in controlling (slowing) the recovery rate upon the reintroduction to a nutrient-rich environment as disassembly of enzyme polymers slows reactivation of the enzyme. Other significant results include the investigation as to whether or not enzyme polymerization serves a purpose in protecting against degradation, and they find that it not, at least in a 24 hour time frame. Interestingly their results also suggestion that some "licensing" process may be required (in addition to low pH) to induce CTPS self-assemblies in cells (a possible example would be phosphorylation). Finally, to bridge the poorly understood connection between enzyme polymerization and enzyme clusters seen in cells, they analyze bundles of polymeric filaments seen in cryo-electron microscopy images. Extrapolating contacts seen in these bundles allowed for the creation of models for large super-structures, which may represent the structure of CTPS in the self-assembly clusters seen in cells, though this is still not yet known. An amino acid insert present in the sequence of yeast CTPS, but not human, is seen to bridge polymeric filaments of CTPS in the bundles but has not yet been investigated using mutagenesis.
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Reviewer #3 (Public Review):
I have only one major issue:
1. Clearly, no study can do everything, but I think the authors should do a bit more to attempt to understand why their "constitutive" assembly mutant does not assemble in vivo in standard conditions.
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