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The intrinsically disordered region from PP2C phosphatases functions as a conserved CO2 sensor

Abstract

Carbon dioxide not only plays a central role in the carbon cycle, but also acts as a crucial signal in living cells. Adaptation to changing CO2 concentrations is critical for all organisms. Conversion of CO2 to HCO3 by carbonic anhydrase and subsequent HCO3-triggered signalling are thought to be important for cellular responses to CO2 (refs. 1,2,3). However, carbonic anhydrases are suggested to transduce a change in CO2 rather than be a direct CO2 sensor4,5, the mechanism(s) by which organisms sense CO2 remain unknown. Here we demonstrate that a unique group of PP2C phosphatases from fungi and plants senses CO2, but not HCO3, to control diverse cellular programmes. Different from other phosphatases, these PP2Cs all have an intrinsically disordered region (IDR). They formed reversible liquid-like droplets through phase separation both in cells and in vitro, and were activated in response to elevated environmental CO2 in an IDR-dependent manner. The IDRs in PP2Cs are characterized by a sequence of polar amino acids enriched in serine/threonine, which provides CO2 responsiveness. CO2-responsive activation of PP2Cs via the serine/threonine-rich IDR-mediated phase separation represents a direct CO2 sensing mechanism and is widely exploited.

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Fig. 1: C. albicans Ptc2 undergoes a reversible phase transition in response to elevated CO2.
Fig. 2: CO2 but not HCO3 directly stimulates Ptc2 activity via IDR-dependent phase separation in fungi.
Fig. 3: Ptc2 controls CO2-responsive W/O switch in C. albicans.
Fig. 4: CO2 sensing via IDR-driven phase transition of PP2Cs is conserved in plants.
Fig. 5: A serine/threonine-rich sequence embedded in IDR of PP2Cs provides CO2 responsiveness.

Data availability

Protein sequence data for proteins CaPtc2, ScPtc2, CnPtc2, AP2C3, PP2C74, Sup35 and FUS that were re-analysed here are available from UniProt under accession codes A0A1D8PRZ8, P39966, F5HBU9, F6LPR7, A0A1P8BC27, P05453 and P35637, respectively. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the members of Lu laboratory for critical discussion and M. Peterson from Liu laboratory for editing the manuscript. We thank S. Sandmeyer and S. Du for strains and plasmids. This work was supported by grants from National Natural Science Foundation of China (31770162 and 32070074 to Y.L., 81973370 and 32170089 to C.S. and 22077094 to C.Z.), a National Key Research and Development Program of China grant (2020YFA0908501 to C.Z.) and a National Institute of General Medical Sciences grant (2R02GM117111-05 to H.L.).

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Authors

Contributions

Y.L., C.S., M.Z. and H.L. designed the experiments. M.Z., Y.D., T.L. and C.S. performed the experiments. M.Z., C.Z., Y.L. and C.S. performed data analyses. Y.L., C.S., M.Z. and H.L. wrote the paper with input from the co-authors.

Corresponding authors

Correspondence to Chang Su or Yang Lu.

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Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Ptc2 protein level is not regulated by CO2 and ectopically expressed Ptc2 cannot promote sustained hyphal development.

a. Western blot analysis of Ptc2-Myc. Wild type cells carrying Ptc2-Myc under its endogenous promoter were incubated in YEPD medium at 30 °C in air or in 5% CO2 for 4 h. b. Overnight cultures of wild type carrying vector or TDH3p-PTC2 were diluted into YEPSucrose medium at 37 °C. Half of the samples was put into the CO2 incubator immediately. The other sample was incubated in air. Cell morphology was examined after incubation of 12 h. The number of cells used for quantification: vector-Air, n=160, 98, 209; vector-CO2, n=105, 92, 106; PTC2-Air, n=119, 139, 128; PTC2-CO2, n=123, 101, 113. The data show the average from three independent experiments. The cells which had a length/width ratio of >4.5 and characteristic shape were considered hyphae. Scale bar, 20 μm.Numerical data and unprocessed blots are available in the source data.

Source data

Extended Data Fig. 2

Sequence identity (%) of the N-terminal PP2C domain and the C-terminal domain of Ptc2 in three indicated yeasts.

Extended Data Fig. 3 CO2 does not significantly affect the saturation concentration of Sup35 and FUS.

a. 5% CO2 significantly decreases the saturation concentration of CaPtc2 whereas pH only marginally influences it. Phase separation of GFP-CaPtc2 under the indicated conditions. Phase separation quantified by the relative amount of condensed protein versus the protein concentration. Images on left were used for the quantification. b. The IDR from Sup35 does not respond to CO2 in driving phase separation. Phase separation of Sup35-GFP and the chimeric construct GFP-Ptc2∆IDR-Sup35IDR under the indicated conditions. Images above were used for the quantification. c. The saturation concentration of FUS does not change significantly in 5% CO2. Phase separation of FUS-GFP and the chimeric construct GFP-Ptc2∆IDR-FUSIDR under the indicated conditions. Images above were used for the quantification. a-c, Scale bar, 5 μm. The saturation concentration is indicated by arrows. Data shown as means ± SD of three independent experiments. Numerical data are available in the source data.

Source data

Extended Data Fig. 4 HCO3- is unable to induce condensates formation of Ptc2 in fungi.

Recombinant GFP-Ptc2 protein droplets formation in the presence of 25 mM NaHCO3. Representative images of three independent experiments are shown. Scale bar, 2 μm.

Extended Data Fig. 5 Ptc2-mediated CO2 sensing mechanism is conserved in fungi.

a. Ptc2 is required for CO2-responsive capsule formation in C. neoformans. Cells of wild type and ptc2 mutant were grown in DME medium in air or 5% CO2 (left and middle column), or in PBS with 10% serum (right column). India ink images were shown after incubation for 6 h. Scale bar, 5 μm. b. Ptc2 controls CO2-responsive carbonic anhydrase expression in yeasts. Exponential cultures of indicated C. albicans, S. cerevisiae and C. neoformans strains were cultivated at 30 °C as duplicates in 5% CO2 for 4 h, and then one duplicate was transferred to air for 60 min to induce carbonic anhydrase expression. The expression was quantified by qRT-PCR analysis and normalized with ACT1. Data shown as means ± SD of three independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. Numerical data are available in the source data.

Source data

Extended Data Fig. 6 Ptc2 regulates CO2-responsive hyphal elongation (a) and carbonic anhydrase expression (b) in an IDR-dependent manner in C. albicans.

Assays of ptc2 carrying PTC2 or PTC2ΔIDR under the control of ADH1promoter were performed as described in Extended Data Fig. 1b and Extended Data Fig. 5b. Scale bar, 20 μm. Data shown in (b) as means ± SD of three independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t-test. Numerical data are available in the source data.

Source data

Extended Data Fig. 7 Coomassie blue staining of purified recombinant proteins used in this study.

Representative gel images of two independent experiments are shown.

Supplementary information

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Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

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Zhang, M., Zhu, C., Duan, Y. et al. The intrinsically disordered region from PP2C phosphatases functions as a conserved CO2 sensor. Nat Cell Biol (2022). https://doi.org/10.1038/s41556-022-00936-6

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