Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana
| Autor: | Yu Ohki, Keisuke Ito, Takeshi Iwatsubo, Tomiko Asakura, Keiko Abe, Taisuke Tomita, Masako Hoshi, Yoshiro Ishimaru |
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| Rok vydání: | 2013 |
| Předmět: |
Proteases
Arabidopsis thaliana Recombinant Fusion Proteins Proteolysis Molecular Sequence Data Saccharomyces cerevisiae Arabidopsis Regulated intramembrane proteolysis (RIP) Biochemistry Substrate Specificity Membrane region medicine Aspartic Acid Endopeptidases Humans Endoplasmic reticulum (ER) Amino Acid Sequence Molecular Biology Peptide sequence medicine.diagnostic_test biology Arabidopsis Proteins Dipeptides biology.organism_classification Aspartic protease Electrophoresis Polyacrylamide Gel Signal peptide peptidase Research Article Signal peptide peptidase (SPP) |
| Zdroj: | BMC Biochemistry |
| ISSN: | 1471-2091 |
| DOI: | 10.1186/1471-2091-14-16 |
| Popis: | Background Signal peptide peptidase (SPP) is a multi-transmembrane aspartic protease involved in intramembrane-regulated proteolysis (RIP). RIP proteases mediate various key life events by releasing bioactive peptides from the plane of the membrane region. We have previously isolated Arabidopsis SPP (AtSPP) and found that this protein is expressed in the ER. An AtSPP-knockout plant was found to be lethal because of abnormal pollen formation; however, there is negligible information describing the physiological function of AtSPP. In this study, we have investigated the proteolytic activity of AtSPP to define the function of SPPs in plants. Results We found that an n-dodecyl-ß-maltoside (DDM)-solubilized membrane fraction from Arabidopsis cells digested the myc-Prolactin-PP-Flag peptide, a human SPP substrate, and this activity was inhibited by (Z-LL)2-ketone, an SPP-specific inhibitor. The proteolytic activities from the membrane fractions solubilized by other detergents were not inhibited by (Z-LL)2-ketone. To confirm the proteolytic activity of AtSPP, the protein was expressed as either a GFP fusion protein or solely AtSPP in yeast. SDS-PAGE analysis showed that migration of the fragments that were cleaved by AtSPP were identical in size to the fragments produced by human SPP using the same substrate. These membrane-expressed proteins digested the substrate in a manner similar to that in Arabidopsis cells. Conclusions The data from the in vitro cell-free assay indicated that the membrane fraction of both Arabidopsis cells and AtSPP recombinantly expressed in yeast actually possessed proteolytic activity for a human SPP substrate. We concluded that plant SPP possesses proteolytic activity and may be involved in RIP. |
| Databáze: | OpenAIRE |
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