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. 2024 Feb 26;36(3):665-687.
doi: 10.1093/plcell/koad289.

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Yong Zou  1 , Igor Sabljić  1 , Natalia Horbach  2 , Adrian N Dauphinee  1 , Anna Åsman  1 , Lucia Sancho Temino  1 , Elena A Minina  1 , Marcin Drag  2 , Simon Stael  1 , Marcin Poreba  2 , Jerry Ståhlberg  1 , Peter V Bozhkov  1
Affiliations

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Yong Zou et al. Plant Cell. .

Abstract

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

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Conflict of interest statement

Conflict of interest statement. None declared.

Figures

Figure 1.
Figure 1.
CrMCA-II is a redox-dependent arginine-specific protease prone to oligomerization. A) Proteolytic activity of rCrMCA-II against AMC-conjugated tetrapeptide substrates with Arg, Lys, or Asp at the P1 position, relative to Ac-VRTR-AMC, under optimal buffer conditions (50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 20 mM CaCl2, 0.1% [w/v] CHAPS, and 7.5 mM DTT). B) Proteolytic activity of rCrMCA-II, as a function of DTT concentration, relative to 100 mM DTT, under optimal buffer conditions, with 50 μM Ac-VRPR-AMC as a substrate. C) Size exclusion chromatography (SEC) analysis of rCrMCA-II in the presence or absence of 1 mM DTT. The insert shows proteolytic activity (relative fluorescence units, RFU) of monomeric and dimeric forms of rCrCMA-II separated in the absence of DTT against Ac-VRPR-AMC under optimal buffer conditions. D) SDS-PAGE analysis of dimer- and monomer-containing fractions from C separated without DTT. The gel was stained with Coomassie Brilliant Blue R250 (CBB). E) Yeast two-hybrid (Y2H) assay of CrMCA-II self-interaction and interaction between the intact (wild-type) protease and its catalytically inactive mutant CrMCA-IIC141A. Interaction between CrMCA-II and serpin, an in vivo inhibitor of plant metacaspases, was used as a positive control. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. The results are a representative example of 2 independent experiments. F) Native PAGE of monomeric and dimeric forms of rCrMCA-II with (+) or without (−) DTT (7.5 mM). The gels were stained with CBB. G) In-gel proteolytic activity assay of monomeric and dimeric forms of CrMCA-II using native PAGE under optimal buffer conditions, with 50 μM Ac-VRPR-AMC as a substrate. * and ** in F) and G) indicate positions of the monomer and dimer, respectively. H) Immunoblot analysis of total protein extracts isolated from the Crmca-ii mutant strain expressing wild-type CrMCA-II tagged with 3×HA (Crmca-ii-23 CrMCA-II-3×HA; see Fig. 3) and fractionated by SEC. Odd fractions from 41 to 91 were separated by SDS-PAGE and analyzed with an anti-HA antibody. The fractions containing protein standards with indicated molecular masses are marked with arrowheads. Based on the calibration curve, the predicted average molecular masses of proteins in fractions 43, 73, and 87 are 1,185 kD, 88.5 kD, and 26.4 kD, respectively. The theoretical masses of CrMCA-II-3×HA zymogen (full-length monomer, FL-3×HA) and C-terminal p10 fragment (p10–3×HA; generated via autocleavage after Arg-190) are 46.1 kD and 25.9 kD, respectively. The fractions enriched for CrMCA-II-3×HA monomers and dimers are denoted by * and **, respectively. I) Relative proteolytic activity of CrMCA-II-3×HA in a subset of SEC fractions from H enriched for megadalton assemblies (fractions 42 to 44), dimers (fractions 72 to 74), monomers (fractions 79 to 81), and a fragment smaller than p10 (fractions 86 to 88) against Ac-VRPR-AMC under optimal buffer conditions, with or without addition of 20 mM CaCl2. Note the presence of a basal level of cell-derived Ca2+ in the assay. The relative VRPRase activity in different SEC fractions was normalized by subtracting background activity caused by proteases other than CrMCA-II-3×HA present in the SEC fractions. This background activity was determined from measuring VRPRase activity in the corresponding SEC fractions obtained from Crmca-ii mutant strain expressing catalytically dead variant of CrMCA-II tagged with 3×HA (strain Crmca-ii-23 CrMCA-IIC141A-3×HA; see Fig. 3 and Supplemental Fig. S7). Data in A), B), and I) represent the means ± standard error of the mean (Sem) of 3 B and I) or 4 A) measurements. Different letters indicate significant differences at P < 0.05, as determined by a 1-way A) or 2-way I) ANOVA with Tukey's honest significant difference test.
Figure 2.
Figure 2.
A chemical toolbox for CrMCA-II. A) HyCoSuL screening data were used to extract optimal peptide sequences for CrMCA-II substrates and activity-based probes. B) Proteolytic activity of recombinant CrMCA-II (rCrMCA-II) against individual fluorescent substrates containing natural (blue bars) or unnatural (red bars) amino acids. Data represent the means ± standard error of the mean (Sem) of triplicate measurements. C) Structures of rCrMCA-II substrates: Ac-VRPR-ACC (commercially available as Ac-VRPR-AMC), Ac-HRTR-ACC (most preferred with natural amino acids), and Ac-H(Bzl)-hS(Bzl)-TR-ACC (most preferred with unnatural amino acids). D) Structure of Cy5-labeled covalent activity-based probe (Cy5-ABP) with an HRTR peptide motif and AOMK electrophilic warhead. E) Binding of different concentrations of Cy5-ABP to 0.4 μM wild-type (WT) rCrMCA-II or its catalytically inactive variant (C141A) under optimal buffer conditions, in the absence or presence of 20 mM CaCl2, as visualized after SDS-PAGE. Oriole staining served as a loading control.
Figure 3.
Figure 3.
Generation of the Crmca-ii mutant and complementation strains in the UVM4 background. A) Diagram of the editing site of CrMCA-II. Red and blue arrows depict pairs of primers used for PCR and quantitative PCR (qPCR) analyses, respectively. The vertical lines in exons 4 and 5 indicate the position of the catalytic dyad of His-87 and Cys-141. B) PCR test using the primer pair depicted in A) with genomic DNA as a template. Distilled water was used as a negative control. Bottom panel shows PCR product for RACK1 (positive control). Crmca-ii-4, Crmca-ii-9, and Crmca-ii-23 are presumed mutant strains. The agarose gel was stained with GelRed. C) qPCR analysis of RACK1, AphvIII, and CrMCA-II copy number using the primer pair depicted in A) and genomic DNA as a template to evaluate genome insertion events. Data represent the means ± standard error of the mean (Sem) of triplicate measurements. Paro, paromomycin resistance cassette. D) Diagram of the CrMCA-II gene structure in the generated strains. The vertical lines indicate the position of the catalytic dyad of His-87 (H) and Cys-141 (C); A represents the Cys-141 for Ala (A) substitution in strains transformed with a catalytically inactive mutant. The dashed vertical lines corresponding to the position of the catalytic dyad indicate abolished transcription of the CrMCA-II gene in the mutant strains. E) Immunoblot analysis of CrMCA-II in UVM4, CrMCA-II mutant (Crmca-ii-9 and Crmca-ii-23), and complementation strains (1 Crmca-ii-23 CrMCA-II-3×HA strain and 2 Crmca-ii-23 CrMCA-IIC141A-3×HA strains [independent colonies from the same transformation event]) using anti-CrMCA-II and anti-HA antibodies. Ponceau staining of the PVDF membrane was used as a loading control. F) Peptide (Ac-HRTR-ACC) cleavage assay of cell lysates (total protein extracts) from the same strains as in E). Data represent the means ± Sem from 2 independent biological experiments, each including 3 replicate measurements.
Figure 4.
Figure 4.
CrMCA-II plays a cytoprotective role during HS. A) Growth of UVM4, Crmca-ii mutants, and complemented strains after HS, as estimated by measuring optical density of liquid cultures at 750 nm (OD750) 6 d after recovery from 2 h of HS at 42 °C. One hundred μL cells at log phase (5 to 6 × 106 cells mL−1) were heat-treated for 2 h at 42 °C. Fifty μL culture was then inoculated into 3 mL fresh TAP medium for recovery and optical density measurements at 750 nm (OD750). Data represent the means ± Sem of 1 representative example of 3 independent experiments, each including 3 or 4 measurements. **P < 0.01, *P < 0.05, as determined by a t-test. B) Percentage of cell death in the UVM4, Crmca-ii mutants, and complemented strains growing under control conditions (23 °C; control) or subjected to 60 min of HS at 42 °C (Heat), as determined by fluorescein diacetate (FDA) staining. Data represent the means ± standard error of the mean (Sem) of 5 independent experiments. Each experiment included 1 or 2 measurements, and each measurement included 600 to 900 cell counts per strain and condition. Different letters indicate significant differences at P < 0.05, as determined by a 2-way ANOVA with Tukey's honest significant difference test. C) Relative CrMCA-II transcript levels in the UVM4 strain at 0, 5, 15, 30, and 60 min into HS at 42 °C, as determined by RT-qPCR. The transcript level at 0 min was set to 1. Data represent the means ± Sem of 3 independent biological experiments. D) Immunoblot analysis of full-length (FL) and the p10 fragment of CrMCA-II in total protein extracts isolated from the UVM4 strain at 0, 5, 15, 30, and 60 min of HS at 42 °C. Ponceau staining of the PVDF membrane was used as a loading control. Data on chart represent means ± Sem of relative abundance of FL CrMCA-II and p10 based on densitometry analysis of the corresponding bands in 4 independent biological experiments. The levels of FL and p10 at 0 min were set to 1. Different letters indicate significant differences at P < 0.05, as determined by a 2-way ANOVA with Tukey's honest significant difference test. E) Relative HRTRase activity of cell lysates isolated from the UVM4 strain at 0, 5, 15, 30, and 60 min into HS at 42 °C. Data represent the means ± Sem of 2 independent biological experiments, each including triplicate measurements. F) Relative HRTRase activity of rCrMCA-II pretreated for 5 min at different temperatures in Ca2+- and DTT-free buffer (nonpermissive conditions) before activity measurements under optimal buffer conditions. Data represent the means ± Sem of triplicate measurements.
Figure 5.
Figure 5.
CrMCA-II localizes to the plasma membrane and modulates its fluidity. A) Plasma membrane (PM) localization of CrMCA-II-mVenus (green) in unstressed UVM4 cells (grown at 23 °C). Cells were stained with FM4-64 (magenta). Scale bar, 10 μm. B) Immunoblot analysis of H+ ATPase and CrMCA-II in different subcellular fractions prepared from unstressed UVM4 cells. LDM, low-density membrane fraction; PM, plasma membrane fraction; EM, endomembrane fraction; Sc, concentrated soluble proteins. Ponceau staining of the PVDF membranes were used as loading controls. FL, full-length CrMCA-II. C) PM-to-cytoplasm translocation of CrMCA-II-mVenus (green) after HS at 42 °C for 60 min. Cells co-expressed mCherry-NLS (turquoise). Scale bar, 10 μm. D) Immunoblot analysis of different subcellular fractions prepared as in B), but after HS at 42 °C for 60 min. E) Diagram showing the separation of the whole cell area into the cytoplasm area (S1) and PM area (S2) for CrMCA-II-mVenus PM-to-cytoplasm signal ratio quantification in F). F) PM-to-cytoplasm ratio of CrMCA-II-mVenus in unstressed (23 °C, control) and heat-stressed (42 °C, heat) cells grown in TAP medium (0.34 mM CaCl2) with or without EGTA (0.75 mM), BAPTA (0.5 mM), or presence of both EGTA (0.75 mM) and CaCl2 (1 mM). ***P < 0.001; **P < 0.01, *P < 0.05, as determined by a t-test. G) Recovery rate of DiOC6(3) fluorescence signal following photobleaching (FRAP) of cells from the indicated strains grown under unstressed conditions (23 °C, control) or HS (39 °C for 60 min, heat). Numbers on the top of the chart indicate fold-increase of PM fluidity in heat-stressed compared to unstressed cells. Different letters indicate significant differences at P < 0.05, as determined by a 2-way ANOVA with Tukey's honest significant difference test. In F) and G), the upper and lower box boundaries respectively represent the first and third quantiles, horizontal lines denote the median, and whiskers indicate the highest and lowest values. Data in F) and G) are from 3 independent biological experiments, each including at least 3 measurements (individual cells).
Figure 6.
Figure 6.
Hypothetical model for CrMCA-II-dependent regulation of thermotolerance. Under favorable growth conditions, CrMCA-II is preferentially present as a zymogen attached to the PM and prevents PM rigidification through an as yet unknown mechanism. HS elevates PM fluidity, the effect of which becomes more dramatic in the Crmca-ii cells with rigidified PM and compromising viability, accounting for their decreased thermotolerance. An increase in PM fluidity under HS stimulates influx of Ca2+ (Schroda et al. 2015) which, in turn, mediates the PM-to-cytoplasm translocation of CrMCA-II. The role of the cytoplasmic pool of CrMCA-II under HS and, in particular, whether it contributes to cytoprotection, remains unknown.
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