Identification and Characterization of SppA, a Novel Light-inducible Chloroplast Protease Complex Associated with Thylakoid Membranes

Abstract

A new component of the chloroplast proteolytic machinery from Arabidopsis thaliana was identified as a SppA-type protease. The sequence of the mature protein, deduced from a full-length cDNA, displays 22% identity to the serine-type protease IV (SppA) from Escherichia coli and 27% identity to Synechocystis SppA1 (sll1703) but lacks the putative transmembrane spanning segments predicted from the E. coli sequence. The N-terminal sequence exhibits typical features of a cleavable chloroplast stroma-targeting sequence. The chloroplast localization of SppA was confirmed by in organello import experiments using an in vitro expression system and by immunodetection with antigen-specific antisera. Subfractionation of intact chloroplasts demonstrated that SppA is associated exclusively with thylakoid membranes, predominantly stroma lamellae, and is a part of some high molecular mass complex of about 270 kDa that exhibits proteolytic activity. Treatments with chaotropic salts and proteases showed that SppA is largely exposed to the stroma but that it behaves as an intrinsic membrane protein that may have an unusual monotopic arrangement in the thylakoids. We demonstrate that SppA is a light-inducible protease and discuss its possible involvement in the light-dependent degradation of antenna and photosystem II complexes that both involve serine-type proteases.Protein degradation is one of the major regulatory processes that allow the adaptation, repair, or removal of thylakoid proteins during environmental or developmental changes (reviewed in Refs. 1 and 2). However, our knowledge about chloroplast proteases remains limited. To date, the components of the degradation machinery that have been identified in the organelle include the stroma-located members of the Clp family, which are well conserved from prokaryotes to eukaryotes (3-7), the stromal (8, 9) and thylakoid (10) processing proteases that dissect imported protein precursors, the thylakoid-bound metalloprotease FtsH (11), and two lumenal components, the heat shock protease HtrA (DegP; Ref. 12) and CtpA, an enzyme involved in the C-terminal processing of the D1 protein (13, 14). However, the actual number of chloroplast-located proteases appears to be much higher as judged from the biochemical purification of numerous chloroplast proteolytic activities in chloroplast subfractions (15) and from the genome analysis of one chloroplast progenic ancestor, the Synechocystis genome, which displays about 40 different protease sequences. Biochemical studies have provided information about substrate and inhibitor specificity for some proteolytic activities (6), but a detailed knowledge of proteolytic processes requires that the enzymes themselves and the corresponding genes are characterized at the molecular level.Two kinds of proteases can be operationally distinguished from a physiological point of view: constitutive proteases that, for instance, are involved in the degradation of abnormal or damaged proteins and inducible proteases that are produced or activated only under c...

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

“…SppA is a light stress-induced, thylakoidbound, ATP-independent Ser-type protease with unknown substrates (Lensch et al, 2001). Interestingly, the very abundant thylakoid FtsH protease complex (FtsH1, FtsH2, FtsH5, and FtsH8;Liu et al, 2010b;Kato et al, 2012), robustly quantified with many MS/ MS spectra (114, 499, 528, and 146 for FtsH1, FtsH2, FtsH5, and FtsH8, respectively; Supplemental Table S3), was not significantly affected (between 0.8-and 1.2-fold).…”

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosusKDDS1

“…There are published reports on a novel light-inducible chloroplast proteinase complex associated with thylakoid membranes, SppA, from Arabidopsis thaliana (a kind of plant, var. Landsberg erecta) 16) and light-dependent chloroplast metalloproteinase complex associated with thylakoid membranes, FtsH, from spinash leaves. 17) SppA is a light-inducible serine-type proteinase participating in the light-dependent turnover of thylakoid membrane proteins or of peptides released from such proteins under light stress conditions.…”

“…17) SppA is a light-inducible serine-type proteinase participating in the light-dependent turnover of thylakoid membrane proteins or of peptides released from such proteins under light stress conditions. 16) FtsH proteinase takes part in degrading proteins whose synthesis and assembly are light-dependent. 17) Hence, the serine proteinase from Rvi.…”

AtFtsH6 is involved in the degradation of the light-harvesting complex II during high-light acclimation and senescence

“…Furthermore, an overexpressed peptide with the N-terminal sequence of pea Lhcb1 has been shown to be degraded in vitro by a stromal serine-type glutamyl endopeptidase and by a cysteinetype enzyme attached to the thylakoid membrane (22). It has also been suggested that the SppA protease might be involved in the degradative regulation of LHC II apoproteins (23). However, the LHC II protease(s) have not been conclusively identified.…”