Yeast-based reporter assay system for identifying the requirements of intramembrane proteolysis by signal peptide peptidase of Arabidopsis thaliana.

FEBS Open Bio
Kenta KusunokiTomiko Asakura

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...Continue Reading

References

May 1, 1990·The Journal of Membrane Biology·G von Heijne
Jan 10, 1998·The EMBO Journal·B MartoglioB Dobberstein
Jun 22, 2002·Science·Andreas WeihofenBruno Martoglio
Nov 7, 2002·Molecular Cell·Marius K Lemberg, Bruno Martoglio
Jul 29, 2006·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Andrew C NyborgTodd E Golde
Aug 3, 2006·The Journal of Biological Chemistry·Paul Targett-AdamsJohn McLauchlan
Apr 22, 2008·The Journal of Biological Chemistry·Paul Targett-AdamsJohn McLauchlan
Jan 27, 2009·Plant Physiology·Sungwon HanDanny J Schnell
May 12, 2009·Seminars in Cell & Developmental Biology·Todd E GoldeDoron C Greenbaum
Sep 22, 2009·Journal of Structural Biology·Xavier DeupiLeonardo Pardo
Dec 14, 2012·Proceedings of the National Academy of Sciences of the United States of America·Michael B HarbutDoron C Greenbaum
May 6, 2015·Trends in Biochemical Sciences·D LangoschM K Lemberg
Apr 4, 2017·European Journal of Cell Biology·Torben MentrupBernd Schröder

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR

Software Mentioned

AtSPP

Related Concepts

Related Feeds