The catalytic and protein-protein interaction domains are required for APM1 function

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Aminopeptidase M1 (APM1) is vital for Arabidopsis thaliana development.
• Mammalian M1 proteases possess distinct enzymatic and protein-protein interaction domains.

Purpose of the Study:

• To investigate the distinct roles of APM1's enzymatic and interaction domains.
• To determine if these domains can function independently in Arabidopsis.

Main Methods:

• Utilized ezetimibe and PAQ-22 inhibitors to probe APM1 functions.
• Generated and analyzed apm1 knockdown and mutant lines.
• Performed complementation studies using human aminopeptidases and other Arabidopsis M1 family members.

Main Results:

• Ezetimibe (inhibitor of protein-protein interactions) and PAQ-22 (inhibitor of catalytic activity) produced distinct phenotypes.
• Catalytically inactive APM1 failed to rescue apm1-1 knockdown mutants.
• APM1 mutants lacking the C-terminus were rescued by catalytically inactive APM1, suggesting interaction domain importance.
• Overexpression of human insulin-responsive aminopeptidase/oxytocinase rescued all apm1 phenotypes, while its catalytically inactive form only rescued the C-terminus mutant.
• APM1 C-terminus is crucial for protein interactions and enzymatic activity facilitation.

Conclusions:

• APM1 requires both catalytic and interaction domains for full function.
• These domains can operate independently and are not required on the same molecular chain for dimerization.
• The C-terminus is critical for APM1's interaction-dependent functions.