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 proteases. Despite common structural fold, caspases and MCAs differ in substrate cleavage specificity and other biochemical properties, including strict requirement of dimerization for activation in caspases and proposed absence thereof in MCAs. Our current structure-function understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis thaliana expressing nine different MCAs with partly redundant and hence difficult to untangle, activities. In contrast to streptophytes, most chlorophyte genomes contain only one or two hitherto uncharacterized MCAs, providing powerful paradigms for evolutionary and mechanistic comprehension of MCAs. Here we investigate a single type II MCA CrMCA-II from a model chlorophyte Chlamydomonas reinhardtii. Unlike other studied MCAs, CrMCA-II dimerizes both in vitro and in vivo. While recombinant CrMCA-II is active in both monomeric and dimeric forms, activation of CrMCA-II in vivo correlates with the dimerization and autocleavage. Thus, the entire pool of CrMCA-II in vivo consists of monomers, dimers and, additionally, large megadalton complexes, with only dimers representing mature protease. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises 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. Finally, we link the non-catalytic 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 evolution of multicellularity.

ATG8 delipidation is not universally critical for autophagy in plants

“…Genomic DNA was isolated as previously described 47 . The cells were grown under standard conditions and harvested at a density of 1-2×10 7 cells ml -1 .…”

“…Total proteins from Chlamydomonas cells were extracted as described in Zou et al, (2023) 47 . The cells were grown under standard conditions and harvested at a density of 1-2×10 7 cells ml -1 .…”