Abstract
Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein (NtPhyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of NtPhyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the NtPhyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein (AtPhyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The AtPhyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from AtPhyt, though the elevated level of intracellular AtPhyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.
Highlights
Phytaspases belong to the vast family of plant subtilisin-like proteases, which includes many members in each plant species, e.g., 56 in Arabidopsis thaliana (Rautengarten et al, 2005), 63 in rice (Oryza sativa, Tripathi and Sowdhamini, 2006), and 82 in grape (Vitis vinifera, Cao et al, 2014; Figueiredo et al, 2016) and tomato (Solanum lycopersicum, Reichardt et al, 2018)
Stress-Induced Internalization of N. tabacum Phytaspase Depends on Clathrin-Mediated Endocytosis and Is Specific
What happens to the proteolytic activity of the enzyme upon internalization? Is the uptake of phytaspases specific, or any soluble apoplastic protein will become internalized upon induction of cell death? Will interference with the phytaspase uptake compromise cell death? Here, we addressed these questions using N. benthamiana leaves either containing endogenous phytaspase only, or overproducing N. tabacum phytaspase (NtPhyt) tagged with EGFP or mRFP that allowed to follow phytaspase re-distribution with the aid of fluorescence microscopy in parallel with the determination of a peculiar Asp-specific activity of phytaspases
Summary
Phytaspases belong to the vast family of plant subtilisin-like proteases (subtilases), which includes many members in each plant species, e.g., 56 in Arabidopsis thaliana (Rautengarten et al, 2005), 63 in rice (Oryza sativa, Tripathi and Sowdhamini, 2006), and 82 in grape (Vitis vinifera, Cao et al, 2014; Figueiredo et al, 2016) and tomato (Solanum lycopersicum, Reichardt et al, 2018). Similar to other plant subtilases, phytaspases are synthesized as proteolytically inactive precursor proteins, which possess an N-terminal signal peptide, a prodomain, and a peptidase domain (Chichkova et al, 2010; Schaller et al, 2018). The precursor protein is autocatalytically and constitutively processed, and the mature proteolytically active enzyme is released into the apoplast (Chichkova et al, 2010), which is typical for plant subtilases. The C-terminal residue of prodomain is Asp, which is consistent with the self-processing mode of generation of the mature enzyme Mutating this junction Asp residue precludes processing/ activation of the phytaspase precursor and the release of the mature enzyme into the apoplast (Chichkova et al, 2010). The number of phytaspase genes appears to vary in plant genomes, from a single gene in A. thaliana to 12 in S. lycopersicum (Chichkova et al, 2018; Reichardt et al, 2018)
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