Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

Abstract

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions. The D1 protein is a membrane-spanning subunit constituting the core part of the photosystem II (PSII) 1 reaction center (1, 2). In the predicted secondary structure, the D1 protein consists of five transmembrane ␣-helices and a C-terminal extension protruding into the luminal space of thylakoids (reviewed in Ref. 2). A curious feature of this protein is the extraordinarily high rate of metabolic turnover in vivo, despite its fundamental importance in the structure and function of PSII (3). In the dynamic process, the D1 protein, which is encoded by the psbA gene in the plastid genome in the case of eukaryotic organisms, is synthesized on membrane-associated ribosomes on the cytosolic or stromal surface of thylakoids in a precursor form with a short C-terminal extension consisting of 8 -16 amino acids (4 -6). This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7-10). In eukaryotic organisms, the enzyme is nuclear encoded (8 -10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e. the manganese cluster in PSII (12-15). However, the absence of this extension by itself at least under the conditions exam...

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

“…The observed transition corresponds to a simple binding isotherm at all wavelengths. The data were fitted to a hyperbolic equation, yielding an apparent K D of ϳ250 M, which is very similar to the K m of the recombinant full D1p (ϳ300 M) (26). This suggests that the isolated D1pPDZ domain binds its physiological target sequence with an affinity consistent with previous experiments.…”

LPA19, a Psb27 Homolog in Arabidopsis thaliana, Facilitates D1 Protein Precursor Processing during PSII Biogenesis

“…The involvement of additional components facilitating the D1 precursor processing has recently been suggested because the recombinant CtpA exhibits a relatively weak affinity toward its substrates in vitro (46). The PratA protein has been implicated in facilitating D1 processing probably through direct interaction with the C-terminal regions.…”

MET1 Is a Thylakoid-Associated TPR Protein Involved in Photosystem II Supercomplex Formation and Repair in Arabidopsis

“…More than a dozen higher plant PSII-specific biogenesis/repair factors have been reported, including HCF136 (Meurer et al, 1998;Covshoff et al, 2008), LPA1 (Peng et al, 2006), FKBP-2 (Lima et al, 2006), CYP38 (Fu et al, 2007;Sirpiö et al, 2008), TLP18.3 (Sirpiö et al, 2007), LPA2 (Ma et al, 2007), LPA3 (Cai et al, 2010), PAM68 (Armbruster et al, 2010), HCF243 (Zhang et al, 2011), LTO1 (Karamoko et al, 2011), TERC (Schneider et al, 2014), LQY1 (Lu et al, 2011), HHL1 (Jin et al, 2014), and psbN (Torabi et al, 2014). Additionally, the lumenal peptidase CtpA is specifically required for C-terminal processing of the D1 protein (Anbudurai et al, 1994;Oelmüller et al, 1996;Yamamoto et al, 2001); in the absence of this C-terminal processing, no active PSII complex can be formed (Che et al, 2013). Thylakoid bound FtsH and Deg proteases play an important role in degrading damaged D1 (Kapri-Pardes et al, 2007;Sun et al, 2010aSun et al, , 2010bChi et al, 2012b;Kato et al, 2012), even if these proteases are not specific to PSII.…”