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

Area of Science:

• Plant molecular biology
• Protease function and regulation
• Cellular stress responses

Background:

• Phytaspases, a unique plant subtilisin-like protease family, exhibit aspartate cleavage specificity and dynamic localization.
• These proteases are secreted by healthy plant cells but re-internalize upon stress perception.

Purpose of the Study:

• To investigate whether the proteolytic activity of phytaspases is essential for their stress-induced retrograde transport.
• To elucidate the mechanism of phytaspase re-localization in response to cellular stress.

Main Methods:

• Utilized an in trans complementation approach in *Nicotiana benthamiana* leaf cells.
• Expressed a prodomain-less phytaspase (NtPhyt) with a free prodomain to achieve active enzyme generation and secretion.
• Generated a catalytically inactive mutant (Ser537Ala) of NtPhyt to assess the role of proteolytic activity in transport.

Main Results:

• Successfully generated proteolytically active NtPhyt and achieved extracellular transport comparable to native phytaspase.
• The inactive NtPhyt Ser537Ala mutant was secreted but retained its ability for retrograde transport upon oxidative stress induction.
• Demonstrated that processed, but inactive, phytaspase can be re-internalized into plant cells.

Conclusions:

• Proteolytic activity of phytaspase is dispensable for its stress-induced retrograde transport.
• The re-internalization mechanism of phytaspase during stress does not rely on its enzymatic function.
• This study clarifies a key aspect of phytaspase localization dynamics and stress response in plants.