RRC ID 64076
Author Kusunoki K, Hoshi M, Tamura T, Maeda T, Abe K, Asakura T.
Title Yeast-based reporter assay system for identifying the requirements of intramembrane proteolysis by signal peptide peptidase of Arabidopsis thaliana.
Journal FEBS Open Bio
Abstract Signal peptide peptidase (SPP) is an aspartic protease with two active sites, YD and GXGD, in the transmembrane domain. SPP cleaves signal peptides, and the released fragments play key roles in the immune system, embryo development and protein turnover in cells. Despite SPP having an important function, a general system to identify the requirements of intramembrane proteolysis by SPP has not been developed because proteolysis occurs in the membrane. In this study, we first established a reporter assay system in yeast to verify the cleavage activity of the Arabidopsis thaliana SPP (AtSPP). Next, we screened candidate substrates of AtSPP from A. thaliana pollen and roots. In the pollen, 13 signal peptides with 'pollen' and 'cell wall' as gene ontology terms were selected. In the roots, mutants overexpressing AtSPP were constructed, and gene expression changes were compared with the wild-type. Nine signal peptides expressed in the roots were selected. Then we used the candidate substrates in our reporter assay system to determine the requirements for proteolysis by AtSPP. Fifteen of 22 signal peptides were cleaved by AtSPP. The absence of the positively charged amino acids, His and Lys on the C terminus of the signal sequence, was observed in cleaved substrates. Moreover, mutation of a helix breaker-to-Leu substitution in the intramembrane region in substrates prevented cleavage by AtSPP. These results indicated that substrates of AtSPP required the helix breaker structure to be cleaved.
Volume 10(9)
Pages 1833-1842
Published 2020-9-1
DOI 10.1002/2211-5463.12936
PMID 32686366
PMC PMC7459403
MeSH Arabidopsis / enzymology* Aspartic Acid Endopeptidases / genetics Aspartic Acid Endopeptidases / metabolism* Proteolysis Saccharomyces cerevisiae / metabolism*
IF 2.231
Arabidopsis / Cultured plant cells, genes pda