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

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 nine MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two 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 a 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.

In vivo proteolytic profiling of the type I and type II metacaspases in Chlamydomonas reinhardtii exposed to salt stress

“…In higher plants, metacaspases are localized mainly in the cytosol and the nucleus, but are also found in mitochondria, the cell wall and the apoplast (Huh, 2022). Recent data suggest the localization of CrMCA-II at the plasma membrane and its translocation to the cytosol during heat stress (Zou et al, 2023). In contrast, in overexpression lines, CrMCA-II was observed in the cytosol during control conditions, while the CrMCA-I signal was restricted at the mitochondria (Lambert et al, 2023).…”

“…In C. reinhardtii, mutant strains of CrMCA-I and CrMCA-II were treated with GSNO to induce oxidative stress and only the CrMCA-II mutant was able to prevent PCD (Lambert et al, 2023). However, in a different study, CrMCA-II mutant cells were shown to be sensitive to heat stress (Zou et al, 2023). Contradictions in the tolerance and viability of the metacaspase mutants denote the complex underlying mechanisms of cellular stress response and the potential multifunctionality of metacaspases.…”

“…The presence of a single copy of these metacaspases in the C. reinhardtii genome, together with the organism's simplicity allowing easily genetic manipulation, make C. reinhardtii an emerging model for metacaspase studies. Recently, two studies have investigated the function of CrMCA-II under diverse settings (Lambert et al, 2023;Zou et al, 2023). CrMCA-II enhanced thermoresistance during heat stress, even in its inactive form (Zou et al, 2023).…”

“…Recently, two studies have investigated the function of CrMCA-II under diverse settings (Lambert et al, 2023;Zou et al, 2023). CrMCA-II enhanced thermoresistance during heat stress, even in its inactive form (Zou et al, 2023). Moreover, CrMCA-II-dependent PCD was documented in response to S-nitrosoglutathione (GSNO) stress (Lambert et al, 2023).…”

Roadmap for the next decade of plant programmed cell death research

“…In turn, the type I metacaspase MC1 participates in clearance of harmful protein aggregates formed as a result of proteotoxic stress, a function conserved from fungi to plants (Lee et al ., 2010; Coll et al ., 2014; Hill et al ., 2014; Ruiz‐Solaní et al ., 2023). On the other hand, both Arabidopsis MC3 and Chlamydomonas CrMCA‐II have been shown to be involved in drought and heat stress tolerance, respectively, independent of their catalytic activity (Pitsili et al ., 2023; Zou et al ., 2023). Collectively, despite the fact that metacaspases may have evolved from the same ancestor as caspases, current evidence indicates that they are not simply executioner caspases in the context of PCD and that they could instead function, or have additional, context‐dependent roles, as pro‐survival proteins.…”