Chloroplast molecular chaperone ClpC in Arabidopsis

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely-related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to the envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to the envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knockout mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import. Molecular chaperones and proteases are integral components of the quality control processes active within the crowded and dynamic protein environment of all living cells. Chaperones are a large diverse group of proteins involved in many structural functions and active cellular processes including protein folding/unfolding and complex assembly/disassembly (1). Certain http://www.jbc.org/cgi/doi/10.1074/jbc.M113.534552 The latest version is at JBC Papers in Press. Published on March 5, 2014 as Manuscript M113.534552 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Chloroplast molecular chaperone ClpC in Arabidopsis 2 chaperones also work in concert with proteases, recognizing polypeptide substrates and unfolding them prior to degradation (2). Proteases themselves are equally crucial to various cellular activities both in maintaining proteostasis and during major cellular events like cell division and differentiation. Chaperones and proteases also perform vital protective and restorative roles during and after periods of stress. Polypeptides that increasingly lose their native structure or become otherwise damaged must be targeted for degradation before they reach cytotoxic levels (3). Clp/Hsp100 proteins are a class of molecular chaperones within the broader family of AAA+ (ATPases Associated with diverse cellular Activities) proteins. They are found in a wide range of organisms from bacteria to mammals and facilitate a diverse array of cellular functions. Most Clp/Hsp100 proteins also function as the regulatory component of Clp proteases. The model Clp protease in E. coli is a two-component enzyme comprised of an oligomeric Hsp100 (either ClpA or ClpX) complex bound to a proteolytic core. The proteolytic core is composed of two opposing heptameric rings of ClpP that together create a central cavity housing the proteolytic active sites (4). The core is flanked on one or both sides by a single hexameric ring of the Hsp100 partner (5). The Hsp100 complex selects substrates and unfolds them, translocating the proteins through the narrow entrance of the Clp proteolytic core into the central chamber (6, 7). Once bound to the active sites, the protein substrate is rapidly degraded to small peptide fragments that eventual diffuse out of the core complex. The substrate specificity of the Hsp100 partner can also be modified by different adaptor proteins such as ClpS that recognize motifs at either the Nor Ctermini of the targeted substrates (8). The Clp protein family in vascular plants is far more diverse than in other organisms, with the majority of these Clp proteins localized in the chloroplast (9). All chloroplast Clp proteins are constitutively expressed and most abundant in leaves compared to other tissues (9, 10). Plant chloroplasts contain four distinct Hsp100 proteins (ClpC1, ClpC2, ClpD and ClpB3) and yet little is still known about their specific chaperone activities or substrate specificity. The closelyrelated ClpC ortholog in the cyanobacterium Synechococcus elongatus has various chaperone characteristics in vitro including the prevention of protein aggregation, and resolubilization/refolding of aggregated polypeptides (11). It also associates to the ClpP3/R proteolytic core to form the principle Clp protease in cyanobacteria and whose function is essential for cell viability (12, 13). A homologous Clp proteolytic core also exists in plant chloroplasts but one that consists of eleven different subunits (14). This core complex comprises two distinct heptameric rings, one with the ClpP3-6 subunits (P-ring) and the other with ClpP1 and ClpR1-4 (R-ring) (15). Also peripherally attached to the P-ring are two accessory proteins ClpT1 and –T2 that are involved in core assembly (16) and possibly substrate recognition (17). All chloroplast Hsp100 proteins with the exception of ClpB3 contain the conserved motifs in the C-terminus necessary for association to the Clp proteolytic core (18). How much the different Hsp100 proteins contribute to the Clp proteolytic activity in chloroplasts, however, remains unknown although a structural association between ClpC and the Clp proteolytic core has been demonstrated (19-21). Genetic studies have clearly shown the crucial role of chloroplast ClpC and the Clp protease for plant viability (15, 22-25). Putative substrates for the chloroplast Clp protease have been identified and range from various metabolic enzymes to regulatory proteins involved in homeostatic functions such as chloroplast gene expression, RNA maturation, protein synthesis and recycling, and tetrapyrrole synthesis (15, 28, 29). Apart from its presumed involvement in Clp proteolysis, ClpC has also been implicated in the chloroplast import of cytosolic preproteins by its close proximity to Tic110, one of the principle subunits of the Tic complex (Translocon at the inner envelope membrane) (30, 31). This together with the finding that ClpC stably binds transit peptides in vitro (32) has suggested that it might function as the driving force for preprotein import through the Tic complex (33). Despite this, evidence for the direct involvement of ClpC in preprotein import and processing in chloroplasts remains elusive. Although ClpC was first shown to be essential for normal chloroplast function many years ago (34), little is still known about its specific characteristics, the interaction between the two paralogs, ClpC1 and ClpC2 or their contribution to Clp proteolytic activity. The amino by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Chloroplast molecular chaperone ClpC in Arabidopsis 3 acid sequences of the two mature ClpC proteins are more than 90% identical (35), and overexpression of ClpC2 can fully complement the loss of ClpC1 in Arabidopsis (25), suggesting both perform similar, if not identical functions in the chloroplast. Mutagenesis studies have shown that loss of ClpC1 in Arabidopsis causes significant phenotypic changes, the most prominent being slower growth rates, leaf chlorosis and impaired photosynthetic activity (36-38), whereas loss of ClpC2 produces little or no effect (37, 39). In this study, we have determined many of the important features of the chloroplast ClpC proteins in Arabidopsis thaliana including their relative abundance, localization and impact upon Clp proteolytic activity. We show that ClpC localized in both the stroma and envelope membrane appears to function in association with the Clp proteolytic core, revealing new dimensions to the functional importance of this essential molecular chaperone in chloroplasts. EXPERIMENTAL PROCEDURES Plant Growth Conditions Seeds of Arabidopsis thaliana wild type (ecotype Columbia-0), and clpC1 knockout (36) and clpC2 knockdown (39) T-DNA insertion lines were sown in a 1:5 perlite/soil mix after vernalization at 4C for at least 48 h. All plants were grown either in individual pots or as lawns under the following standard conditions: 8 h photoperiod with white light (ca. 150 μmol photons m s), 23C/18C day-night temperatures and 65% relative air humidity. Immunoblotting from leaf protein extracts Total cell proteins were isolated from leaves from wild type, clpC1 and clpC2 mutant plants as previously described (15). The protein concentration of each final sample extract was determined using the Bradford protein assay (BioRad). Samples were then loaded, based on equal protein content, on pre-cast 3-8% Tris-acetate NuPAGE gels (Invitrogen). Following electrophoretic separation, proteins were transferred to supported nitrocellulose (Bio-Rad) using an Xcell blotting apparatus (Invitrogen). For detecting the different Clp proteins, specific polyclonal antibodies were used as previously detailed (16, 36, 39). Primary antibodies were detected with the horseradish peroxidase-linked, anti-rabbit IgG secondary antibody from donkey and visualized by enhanced chemiluminescence (ECL Advance, GE Healthcare). Chemiluminescent signals were detected and quantified using the ChemiGenius imaging system (Syngene) and associated software. To ensure correct loading, samples were separated on additional gels and stained with coomassie brilliant blue G-250 to detect the relative amount of Rubisco large subunit (LSU). Fractionation of stromal, thylakoid membrane and envelope membrane proteins Intact chloroplasts were isolated from wild type Arabidopsis, and clpC1 and clpC2 mutant plants according to Sjögren et al. (15). Fractionation of stromal, thylakoid membrane and envelope membrane proteins was performed as previously described (40). The protein concentration of the stroma and envelope membrane fractions was determined using the Bradford protein assay (BioRad), whereas the Chl concentration of the leaf and thylakoid membrane fraction was measured according to Porra et al. (41). Samples from each fraction along with one from whole leaves were separated on pre-cast 3-8% Trisacetate gels or linear 12% Bis-Tris gels NuPAGE gels (Invitrogen) depending on the size range of the proteins being examined. Following separation, proteins were transferred to a supported nitrocellulose membrane (Bio-Rad) using an Xcell blotting apparatus (Invitrogen). Amounts of ClpC1, ClpC2, total ClpC, ClpD and ClpP6 were then determined by immunoblotting with specific antibodies as described above, as were the control proteins for the stromal (small subunit of Rubisco, SSU), envelope membrane (Tic110) and thylakoid membrane (Lhcb2) fractions. Detection and quantification of antibody signals were done as described above. Protein-complex separation by CN-PAGE Protein complexes from stromal fractions were separated by colorless native-PAGE (CN-PAGE) as previously detailed (16). After separation, all proteins were transferred to supported nitrocellulose using a semi-dry electrophoretic cell (Trans-Blot, Bio-Rad). The Clp proteolytic core (325 kD) was detected using antibodies specific for three different subunits of the core complex ClpP6, ClpR2 and ClpT1 (15). Detection and quantification of antibody signals were done as described above. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Chloroplast molecular chaperone ClpC in Arabidopsis 4 Protein-complex separation by BN-PAGE Protein complexes from the envelope-membrane fraction were separated by blue native-PAGE (BNPAGE) as previously described (42) with the following modifications. The envelope membrane sample was pelleted by centrifugation at 130,000 g for 30 min and then solubilized in 25 mM BisTris/HCl pH 7.5, 1.5% (w/v) n-dodecyl β-Dmaltoside, 20% (w/v) glycerol and 0.5 M aminocaproic acid. The sample was incubated on ice for 10 min and then centrifuged at 20,000 g for 20 min to pellet any residual insoluble material. Prior to loading, 0.1 volumes of sample buffer (100 mM BisTris/HCl pH 7.5, 0.5 M aminocaproic acid, 30% (w/v) sucrose, and 50 mg/ml Briliant Serva Blue G-250) was added to both stroma and envelope membrane samples. Samples were separated on 4-13% polyacrylamide gradient gels as previously described (16, 42) and then transferred to supported nitrocellulose using a semi-dry electrophoretic cell (Trans-Blot, BioRad). The Clp proteolytic core and subcomplexes (Pand R-rings) were detected using antibodies specific for different subunits – ClpP4, ClpP6, ClpR3 and ClpR4 (15). Detection and quantification of antibody signals were done as described above. Purification of recombinant Clp proteins The Arabidopsis CLPP4 and CLPP5 gene sequences (excluding the region coding for the chloroplast transit peptide) were commercially synthesized (Invitrogen), with the codon sequences optimized for E. coli protein overexpression. Restriction sites were also included at the 5’ and 3’ ends of both genes (NcoI/BamHI for CLPP4, NdeI/KpnI for CLPP5) to facilitate cloning into the pACYC Duet expression vector (Novagen), along with the sequence for a His6-tag at the 3’ end to aid purification. The ClpP4 and ClpP5 proteins were over-expressed in E. coli BL21-STAR cells (Invitrogen) and purified by sequential affinity and gel filtration chromatography as previously described (11). The purified ClpP4 and ClpP5 were stored in 20 mM Tris/HCl pH 7.5, 75 mM NaCl, 1 mM DTT and 20% glycerol (w/v). The DNA sequence coding for the mature Arabidopsis ClpD protein was PCR amplified from a full-length cDNA clone and ligated into the pCDF expression vector (Novagen). Restriction sites NcoI and NotI were included at the 5’ and 3’ ends, respectively to facilitate cloning along with a His6 tag at the 3’ end to enable later protein purification. The ClpD protein was over-expressed in E. coli BL21-CodonPlus (Stratagene) as previously described (11), but formed inclusion bodies that were resolubilized in 6M urea, 20 mM Tris/Cl pH 8, 300 mM NaCl, 40 mM imidazole and 0.5 mM DTT. The soluble ClpD was then purified by sequential Ni affinity and gel filtration chromatography according to Andersson et al. (11). Recombinant Synechococcus ClpC was purified as previously described (11). Chloroplast protein import experiments Chloroplasts from 14-d-old plants were isolated according to Aronsson and Jarvis (43, 44) and used for the import assays. Template DNA from Arabidopsis cDNA clones for the precursors of the Rubisco small subunit (pSS), the subunit II of CF0 of the photosynthetic ATPase (pCFoII), and plastocyanin (pPC) were amplified using M13 primers and used for in vitro transcription /translation using a coupled TNT system (Promega) based on rabbit reticulocyte lysate containing [35S]-methionine and T7 RNA polymerase (43, 44). Chloroplast protein import reactions were performed as described by Aronsson and Jarvis (43, 44). Briefly, each 200 μl import assay contained 10 chloroplasts, 5 mM MgATP, translation mixture not exceeding 10% of the total volume, and was carried out in white light (100 μmol photons m sec) at 25oC for various time periods. Samples were resolved on 12% SDS–PAGE gels. The gels were fixed, exposed to X-ray film, and then quantified using ImageQuant software (Molecular Dynamics). Protein Degradation Assay Intact chloroplasts from wild type, clpC1 and clpC2 mutant plants were isolated as previously described (15). The number of intact chloroplasts in each preparation was determined by phase contrast microscopy (Olympus BX50) using a haemocytometer. Each intact chloroplast sample was diluted to a final concentration of 1.5 × 10 chloroplasts μl with 5 mM Mg-ATP, 2.5 mM phosphocreatine, 50 mg ml creatine phosphokinase, 0.33 M sorbitol, 5 mM MgCl2, 10 mM NaHCO3 and 20 mM HEPES/NaOH pH 8. Chloroplasts were incubated for 3 h under white light (ca. 60 μmol photons m s) at 25C. Reactions were stopped by adding five volumes of rupture buffer (10 mM MgCl2, 20 mM by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Chloroplast molecular chaperone ClpC in Arabidopsis 5 HEPES/NaOH pH 7.6), and then centrifuged at 20,000g for 10 min to separate the thylakoid membranes from the stroma/envelope membrane fraction. Protein concentration of the stromal/envelope membrane fraction was determined using the BCA protein assay (Pierce Chemicals). Protein samples were separated by denaturing-PAGE on either 3-8% Tris-acetate (for EF-Ts, HSP90, RH3 and Tic110) or 12% Bis-Tris (for Tic55, Tic40 and Tic20) gels. The amount of each protein substrate was detected by either staining with coomassie brilliant blue G-250 (EFTs, HSP90 and RH3) or by immunoblotting using a specific antibody (Tic110, Tic40, Tic20 and Tic55). Quantifications were performed using the ChemiGenius imaging system (Syngene) and associated software.