Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

Abstract

BackgroundSignal 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.ResultsWe 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.ConclusionsThe 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.

Arabidopsis thaliana intramembrane proteases

“…Many studies also indicate that intramembrane protease performs crucial functions in providing plant fertility. Aberrations in flower, pollen or silique development were observed in rbl10 and atkom mutants as well as in spp (Thompson et al 2012, Hoshi et al 2013. The transcripts of SPPL1, SPPL2 and SPPL3 proteins accumulate in turn during seed germination, but their role in this process remains unexplored.…”

“…Continuously accumulating data indicate; however, that their role in development should not be underestimated. A mutation in the SPP gene was demonstrated to be lethal (Hoshi et al 2013). Many studies also indicate that intramembrane protease performs crucial functions in providing plant fertility.…”

“…The interesting fact is that this effect was reversed by the expression of both active and inactive presenilin variants, suggesting that the protease acts independently from the c-secretase complex (Khandelwal et al 2007). SPP is a proteolytically active protein crucial for A. thaliana development, since knockout of the SPP gene resulted in a lethal phenotype (Han et al 2009, Hoshi et al 2013. Studies performed on spp heterozygotes indicate that the protein is involved in pollen development and germination (Hoshi et al 2013).…”

“…SPP is a proteolytically active protein crucial for A. thaliana development, since knockout of the SPP gene resulted in a lethal phenotype (Han et al 2009, Hoshi et al 2013. Studies performed on spp heterozygotes indicate that the protein is involved in pollen development and germination (Hoshi et al 2013). The SPPL1, SPPL2 and SPPL3 transcripts were detected in the tissues, roots, rosette leaves, cauline leaves, stems, flower-bud clusters, siliques and dry seeds (Han et al 2009) The significant accumulation of all three transcripts was; however, observed during seed germination.…”

Membrane-Bound Transcription Factors in Plants: Physiological Roles and Mechanisms of Action

“…Four families of multispanning membrane proteases, S2P, rhomboid, presenilin, and signal peptide peptidase (SPP), have been well characterized in animals. Substrates of two other multispanning membrane proteases, presenilin, and SPP (Smolarkiewicz et al, 2014;Han et al, 2009;Hoshi et al, 2013;Tamura et al, 2009), remain to be identified in plants. Rhomboid is encoded by multiple genes in the Arabidopsis genome (Kanaoka et al, 2005) and is proposed to be required for proteolytic cleavage of ANAc017 as described in Section 25.3.5.…”