Aspartic proteases from Silybum marianum: different plant-specific inserts, different destinations

Area of Science:

• Plant molecular biology
• Enzymology
• Protein trafficking

Background:

• Typical plant aspartic peptidases (APs) possess a unique saposin-like domain called the plant-specific insert (PSI).
• The PSI is hypothesized to play a role in directing APs to the vacuole.
• The precise mechanisms by which PSIs influence protein trafficking remain incompletely understood.

Purpose of the Study:

• To clone and characterize two novel typical APs, AP-Sm1 and AP-Sm2, from Silybum marianum flowers.
• To investigate the structural features and potential functional differences of their plant-specific inserts (PSIs).
• To elucidate the role of PSIs and glycosylation in the subcellular localization of these plant aspartic peptidases (APs).

Main Methods:

• In silico analysis and phylogenetic comparisons of novel AP sequences.
• Cloning and characterization of AP-Sm1 and AP-Sm2 from Silybum marianum.
• Subcellular localization studies using mRFP-fused proteins and analysis of vacuolar sorting determinants.

Main Results:

• Two novel typical aspartic peptidases (APs), AP-Sm1 and AP-Sm2, were identified with distinct plant-specific inserts (PSIs).
• AP-Sm1 exhibited glycosylation within its PSI, while AP-Sm2 did not show this pattern.
• Differential subcellular localization was observed: AP-Sm2 localized to the vacuole, but AP-Sm1 was found in the apoplast when the C-terminal vacuolar sorting determinant was non-functional.

Conclusions:

• Plant-specific inserts (PSIs) and their associated glycosylation patterns significantly influence the intracellular trafficking of typical aspartic peptidases (APs).
• Variations in PSI structure and modification can lead to differential localization, such as vacuolar versus apoplastic targeting.
• These findings establish AP PSIs as valuable models for studying protein trafficking in plant cells.