Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of phytaspase (Phyt) with the free prodomain in leaf cells. Using this approach, the generation of the proteolytically active Phyt and its transport to the extracellular space at a level comparable to that of the native Phyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to Phyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized Phyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of Phyt is dispensable for stress-induced retrograde transport of the enzyme.