NblA, a Key Protein of Phycobilisome Degradation, Interacts with ClpC, a HSP100 Chaperone Partner of a Cyanobacterial Clp Protease
2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês
10.1074/jbc.m805823200
ISSN1083-351X
AutoresAnne Karradt, Johanna Sobanski, Jens Mattow, Wolfgang Lockau, Kerstin Baier,
Tópico(s)Plant Stress Responses and Tolerance
ResumoWhen cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the α-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC·ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process. When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the α-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC·ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process. Nitrogen deficiency causes a dramatic color change of cyanobacterial cultures from blue-green to yellow-green, a phenomenon described as “nitrogen chlorosis” nearly 100 years ago (1Boresch K. Lotos (Prague). 1910; 58: 344-345Google Scholar). This bleaching is mainly due to the degradation of the cyanobacterial antenna pigment complexes called phycobilisomes (2Allen M.M. Smith A.J. Arch. Mikrobiol. 1969; 69: 114-120Crossref PubMed Scopus (297) Google Scholar, 3Lau R.H. MacKenzie M.M. Doolittle W.F. J. Bacteriol. 1977; 132: 771-778Crossref PubMed Google Scholar). Phycobilisomes (PBS) 2The abbreviations used are: PBS, phycobilisome; Nostoc 7120, Nostoc sp. PCC7120; BaClpC, ClpC of Bacillus subtilis; PCC, Pasteur Culture Collection; PC, phycocyanin; PEC, phycoerythrocyanin; SyClpC, ClpC of Synechococcus elongatus PCC7942; Synechococcus 7942, Synechococcus elongatus PCC7942; GST, glutathione S-transferase; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 2The abbreviations used are: PBS, phycobilisome; Nostoc 7120, Nostoc sp. PCC7120; BaClpC, ClpC of Bacillus subtilis; PCC, Pasteur Culture Collection; PC, phycocyanin; PEC, phycoerythrocyanin; SyClpC, ClpC of Synechococcus elongatus PCC7942; Synechococcus 7942, Synechococcus elongatus PCC7942; GST, glutathione S-transferase; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. are giant protein complexes consisting of chromophorylated phycobiliproteins and non-pigmented linker peptides. Each PBS is composed of a core, which is anchored to the thylakoid membrane, and peripheral rods building the typical hemidiscoidal structure (4Glazer A.N. Annu. Rev. Microbiol. 1982; 36: 173-198Crossref PubMed Scopus (181) Google Scholar, 5MacColl R. J. Struct. Biol. 1998; 124: 311-334Crossref PubMed Scopus (504) Google Scholar, 6Adir N. Photosyn. Res. 2005; 85: 15-32Crossref PubMed Scopus (249) Google Scholar). Phycobiliproteins can constitute up to half of the total soluble protein of a cyanobacterial cell (7Bogorad L. Annu. Rev. Plant Physiol. 1975; 26: 369-401Crossref Google Scholar). Their degradation during nitrogen deprivation is part of a complex acclimation process that is thought to fulfill two functions: preventing photo damage under stress and providing substrates for synthesis of proteins required for the adaptation. PBS degradation proceeds in an ordered manner, beginning with the successive loss of the peripheral rods (trimming) and ending with the degradation of the core components (8Yamanaka Y. Glazer A.N. Arch. Microbiol. 1980; 124: 39-47Crossref Scopus (129) Google Scholar, 9Collier J.L. Grossman A.R. J. Bacteriol. 1992; 174: 4718-4726Crossref PubMed Scopus (227) Google Scholar). It is reversible at any time. As soon as a nitrogen source is available again, the PBS are resynthesized. The detailed mechanism of PBS degradation is not yet understood. However, screening for non-bleaching (nbl) mutants of Synechococcus elongatus PCC7942 (Synechococcus 7942) revealed several genes that are involved in the degradation process (10Collier J.L. Grossman A.R. EMBO J. 1994; 13: 1039-1047Crossref PubMed Scopus (200) Google Scholar, 11Schwarz R. Grossman A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11008-11013Crossref PubMed Scopus (122) Google Scholar, 12Dolganov N. Grossman A.R. J. Bacteriol. 1999; 181: 610-617Crossref PubMed Google Scholar, 13van Waasbergen L.G. Dolganov N. Grossman A.R. J. Bacteriol. 2002; 184: 2481-2490Crossref PubMed Scopus (116) Google Scholar, 14Sendersky E. Lahmi R. Shaltiel J. Perelman A. Schwarz R. Mol. Microbiol. 2005; 58: 659-668Crossref PubMed Scopus (29) Google Scholar). Among the proteins encoded by these “non-bleaching” genes, one (nblA encoding protein NblA) is directly involved in PBS degradation and appears to play the key role in this process (10Collier J.L. Grossman A.R. EMBO J. 1994; 13: 1039-1047Crossref PubMed Scopus (200) Google Scholar). Inactivation of orthologs of nblA led to non-bleaching phenotypes in several other cyanobacterial species (15Baier K. Nicklisch S. Grundner C. Reinecke J. Lockau W. FEMS Microbiol. Lett. 2001; 195: 35-39Crossref PubMed Google Scholar, 16Li H. Sherman L.A. Arch. Microbiol. 2002; 178: 256-266Crossref PubMed Scopus (59) Google Scholar, 17Baier K. Lehmann H. Stephan D.P. Lockau W. Microbiology. 2004; 150: 2739-2749Crossref PubMed Scopus (61) Google Scholar). In the filamentous, diazotrophic cyanobacteria Nostoc sp. PCC7120 (Nostoc 7120) and Anabaena variabilis ATCC 29413 this non-bleaching phenotype is most obvious in heterocysts, cells specialized for fixing N2 under aerobic conditions, which usually contain only low levels of phycobiliproteins (18Wolk C.P. Bacteriol. Rev. 1973; 37: 32-101Crossref PubMed Google Scholar, 19Meeks J.C. Elhai J. Microbiol. Mol. Biol. Rev. 2002; 66: 94-121Crossref PubMed Scopus (298) Google Scholar). Upon nitrogen step-down, transcription of nblA is highly up-regulated (10Collier J.L. Grossman A.R. EMBO J. 1994; 13: 1039-1047Crossref PubMed Scopus (200) Google Scholar, 15Baier K. Nicklisch S. Grundner C. Reinecke J. Lockau W. FEMS Microbiol. Lett. 2001; 195: 35-39Crossref PubMed Google Scholar, 16Li H. Sherman L.A. Arch. Microbiol. 2002; 178: 256-266Crossref PubMed Scopus (59) Google Scholar, 17Baier K. Lehmann H. Stephan D.P. Lockau W. Microbiology. 2004; 150: 2739-2749Crossref PubMed Scopus (61) Google Scholar, 20Luque I. Zabulon G. Contreras A. Houmard J. Mol. Microbiol. 2001; 41: 937-947Crossref PubMed Scopus (60) Google Scholar, 21Richaud C. Zabulon G. Joder A. Thomas J.-C. J. Bacteriol. 2001; 183: 2989-2994Crossref PubMed Scopus (100) Google Scholar, 22Luque I. Ochoa de Alda J.A. Richaud C. Zabulon G. Thomas J.-C. Houmard J. Mol. Microbiol. 2003; 50: 1043-1054Crossref PubMed Scopus (33) Google Scholar). Increase of nblA mRNA is considered as characteristic for nitrogen deprivation. Genes homologous to nblA are present in most phycobiliprotein-containing cyanobacteria and in red algae. Of the 26 nblA orthologs found in databases, 20 are from cyanobacterial strains, 5 from chloroplasts of red algae, and 1 from a cyanophage. However, nblA seems to be absent from Prochlorococcus strains, from marine Synechococcus subclusters MC-A and MC-B, 3M. Ostrowski, Dept. of Biological Sciences, University of Warwick, personal communication. 3M. Ostrowski, Dept. of Biological Sciences, University of Warwick, personal communication. and from Gloeobacter violaceus PCC7421 (24Nakamura Y. Kaneko T. Sato S. Mimuro M. Miyashita H. Tsuchiya T. Sasamoto S. Watanabe A. Kawashima K. Kishida Y. Kiyokawa C. Kohara M. Matsumoto M. Matsuno A. Nakazaki N. Shimpo S. Takeuchi C. Yamada M. Tabata S. DNA Res. 2003; 10: 137-145Crossref PubMed Scopus (216) Google Scholar). The NblA proteins are rather small, consisting of around 60 amino acid residues corresponding to a molecular mass of ∼7 kDa. Although the homology between different NblA sequences is not very high (about 30% sequence identity on average), they seem to share similar structures. The crystal structure of NblA from Nostoc 7120 demonstrates that the NblA polypeptide consists of two α-helices, a shorter N-terminal and a longer C-terminal one. Two NblA molecules form a homodimer in which the C-terminal helices are involved in dimerization (25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Crystallographic data of NblA from Thermosynechococcus vulcanus suggest a similar structure for this protein (26Dines M. Sendersky E. Schwarz R. Adir N. J. Struct. Biol. 2007; 158: 116-121Crossref PubMed Scopus (8) Google Scholar). Sequence and structure of NblA show no significant similarity to proteins with known function, and the molecular mechanism of NblA action in PBS degradation is not clear. In vitro studies showed that NblA interacts with the α-subunits of phycobiliproteins (22Luque I. Ochoa de Alda J.A. Richaud C. Zabulon G. Thomas J.-C. Houmard J. Mol. Microbiol. 2003; 50: 1043-1054Crossref PubMed Scopus (33) Google Scholar, 25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Furthermore, results of binding experiments with variants of NblA carrying amino acid substitutions at various positions provided evidence that the interaction with phycobiliproteins is mediated via conserved amino acid residues near its C terminus (25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, the most highly conserved stretch of amino acids of the protein is located near its N terminus. In this study we show that NblA interacts with a chaperone, the HSP100/Clp protein ClpC, via a highly conserved motif located at the beginning of its N-terminal helix. Bacterial Strains and Growth Conditions—Strains are listed in supplemental Table S1. Nostoc 7120 and its mutants were grown at 28 °C under constant illumination (cool-white fluorescent lamps, average light intensity 50 μmol of photon m–2 s–1) in BG11 medium that contains 17.6 mm sodium nitrate as nitrogen source, or in BG110, which lacks sodium nitrate (27Rippka R. Methods Enzymol. 1988; 167: 3-27Crossref PubMed Scopus (653) Google Scholar). Liquid cultures were bubbled with 2% (v/v) CO2 in air. For growth on plates, the medium was solidified with separately autoclaved 0.6% (w/v) agar (Difco Laboratories, Detroit, MI). Mutants were grown in the presence of 150 μg ml–1 neomycin, 4 μg ml–1 spectinomycin, and 1 μg ml–1 streptomycin, respectively. For nitrogen starvation experiments, cyanobacterial cultures exponentially growing in BG11 medium were collected by filtration through a nylon net filter (11 μm pore size, Millipore Corporation, Bedford, MA), washed twice with BG110, resuspended in this medium to a concentration of about 5 μg of chlorophyll ml–1 and grown further. Chlorophyll content was estimated in methanolic extracts according to Tandeau de Marsac and Houmard (28Tandeau de Marsac N. Houmard J. Methods Enzymol. 1988; 167: 318-328Crossref Scopus (263) Google Scholar). Whole cell absorbance spectra were recorded in the wavelength range from 550 to 750 nm on a Shimadzu UV-2401PC spectrophotometer equipped with an integrating sphere and were corrected for residual cell scattering at 750 nm. Strains of Escherichia coli (E. coli) were grown under standard conditions. When appropriate, antibiotics were added to the medium to final concentrations of 50 μg ml–1 ampicillin, 50 μg ml–1 kanamycin sulfate, 25 μg ml–1 chloramphenicol, or 50 μg ml–1 spectinomycin, respectively. Plasmid and Mutant Constructions—Plasmids and oligonucleotides are listed in supplemental Table S1. For reversion of the Nostoc 7120 ΔnblA mutant to the wild-type phenotype, a 1239-bp fragment, bearing the chromosomal nblA gene asr4517 (kazusa.or.jp/cyano/Nostoc/index.html) (29Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Shimpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 205-213Crossref PubMed Scopus (576) Google Scholar) together with upstream and downstream sequences, was amplified by PCR using primers 2.11 (XbaI site inserted) and 2.12 (XhoI site inserted), and 2.16 (XhoI site inserted) and 2.17 (EcoRV and BglII site inserted), respectively. The fragments were ligated into the pIC20R vector (73Marsh J.L. Erfle M. Wykes E.J. Gene (Amst.). 1984; 32: 481-485Crossref PubMed Scopus (522) Google Scholar) to yield plasmid pIC20RnblA-T. For details of construction see supplemental Fig. S1A. Using plasmid pIC20RnblA-T as template, primers were designed to produce site-specific mutations, and mutagenesis was performed using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). All DNA constructs were confirmed by DNA sequencing. The fragments harboring the nblA gene or its mutated variants, flanked by the promoter and terminator regions, were then excised by EcoRI digestion and ligated into the self-replicating plasmid pRL1049 (74Black T.A. Wolk C.P. J. Bacteriol. 1994; 176: 2282-2292Crossref PubMed Scopus (135) Google Scholar). Transfer of plasmids between the ΔnblA mutant of Nostoc 7120 and E. coli was achieved by conjugation using E. coli strain J53 bearing RP4 and cargo strain HB101, bearing helper plasmid pRL528, in triparental matings (30Elhai J. Wolk C.P. Methods Enzymol. 1988; 167: 747-754Crossref PubMed Scopus (434) Google Scholar). Exconjugants were selected on BG11 agar plates containing 150 μg ml–1 neomycin, 4 μg ml–1 spectinomycin, and 1 μg ml–1 streptomycin. Expression of NblA-GST Fusion Protein in Nostoc 7120—For expression of NblA C-terminal fused to GST in ΔnblA mutant cells the nblA gene together with upstream sequences was amplified by PCR using primers 2.11 (XbaI site inserted) and 2.12 (XhoI site inserted) and ligated into the pIC20R vector yielding plasmid pIC20RnblA. This pIC20RnblA vector was used to create an NdeI site at the 3′ end of the nblA coding region. The NdeI site was generated by mutagenesis (QuikChange® site-directed mutagenesis kit; Stratagene) using plasmid pIC20RnblA as template and the primers QCM23for and QCM24rev. This mutagenesis resulted in the change from Thr63 to a histidine residue and the loss of the two amino acids, Pro64 and Ala65, of the NblA protein. The resulting plasmid pIC20RnblA/NdeI was digested with BglII and NdeI (partial digestion) and subsequently ligated to a fragment harboring the glutathione S-transferase (gst) gene excised from plasmid pGEX-2TK/NdeI (see “Construction of Plasmids for Expression of GST Fusion Proteins”) by NdeI and BamHI digestion. The resultant construct was restricted with EcoRV and, after dephosphorylation, ligated to a PCR fragment bearing the terminator region of the nblA gene, amplified using primers 2.17 (EcoRV and BglII site inserted) and 2.20 (EcoRV site inserted). For schematic representation of construction see supplemental Fig. S1B. Plasmid pIC20RnblAgst-T was verified by DNA sequencing. Restriction with EcoRI yielded a fragment encoding the GST-tagged NblA protein with promoter and terminator regions that was ligated into the self-replicating plasmid pRL1049. The transfer of plasmids between the ΔnblA mutant of Nostoc 7120 and E. coli was attained as described above. Purification of the NblA-GST Fusion Protein from Nostoc 7120—Cultures of the ΔnblA mutant expressing the GST-tagged NblA protein, transferred to BG110 medium (which lacks an N-source) for 6, 8, and 15.5 h, respectively, were harvested by filtration through a nylon net filter (11 μm pore size, Millipore Corporation, Bedford, MA). The three pellets (each containing ∼8 mg of chlorophyll) were collected and resuspended in ∼30 ml of sodium/potassium phosphate buffer (20 mm sodium/potassium phosphate (pH 7.6), 200 mm KCl, 20 mm NaCl, 2 mm MgCl2, 10% (v/v) glycerol). The following steps were performed at 4 °C. Cells were disrupted in a bead beater (Hamilton Beach) using glass beads of 0.25–0.5 mm. Glass beads and unbroken cells were removed by centrifugation for 5 min at 4,000 × g. The insoluble fraction consisting of cells, cell fragments, and membranes was removed by two centrifugations (1.5 h, 164,000 × g) and the supernatant incubated with ∼150 μl of Glutathione-Sepharose™ 4B (GE Healthcare) in sodium/potassium phosphate buffer overnight at 4 °C with slow agitation. Crude extracts of the ΔnblA mutant expressing the non-tagged NblA served as control. After washing with sodium/potassium phosphate buffer (2 times, 800 μl each), bound proteins were eluted 3 times with 200 μl of 40 mm glutathione in 50 mm Tris/HCl (pH 8.0). Eluates were concentrated by using Vivaspin 500 tubes (Membrane: 5,000 MWCO, Vivascience, New York) and analyzed by SDS-PAGE. SDS-PAGE was performed in slab gels containing 12% (w/v) acrylamide:methylene bisacrylamide (29:1) in the buffer system of Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Following electrophoresis, protein bands were visualized by colloidal Coomassie Brilliant Blue G-250 staining (32Doherty N.S. Littman B.H. Reilly K. Swindell A.C. Buss J.M. Anderson N.L. Electrophoresis. 1998; 19: 355-363Crossref PubMed Scopus (159) Google Scholar). For protein identification, protein bands of interest were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting as well as by MALDI-MS/MS (33Mattow J. Siejak F. Hagens K. Schmidt F. Köhler C. Treumann A. Schaible U.E. Kaufmann S.H. Proteomics. 2007; 7: 1687-1701Crossref PubMed Scopus (28) Google Scholar). To this end, protein bands were excised from Coomassie Brilliant Blue G-250-stained gels and in-gel digested using porcine sequencing grade modified trypsin (Promega, Madison, WI). The resultant proteolytic peptides were desalted and concentrated using ZipTipμ-C18 pipette tips (Millipore Corporation, Bedford, MA) and analyzed with a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) using α-cyano-4-hydroxy-cinnamic acid (Sigma) as a matrix. The MS spectra were recorded in the reflectron mode of the 4700 Proteomics Analyzer and calibrated using trypsin autolysis peaks as internal markers. The MS/MS spectra were acquired without applying collision-induced dissociation. The spectra were processed using the 4700 Explorer software version 3.0 (Applied Biosystems, Foster City, CA). The peak lists of the spectra were created by the Peak-to-Mascot script of the 4700 Explorer software using the following filter settings: mass range from 500 to 4,000 Da (MS); 60 Da to precursor mass −20 Da (MS/MS); peak density, ≤10 peaks per 200 Da (MS), ≤20 peaks per 100 Da (MS/MS); minimal signal-to-noise-ratio, 20 (MS), 3 (MS/MS); minimal peak area, 1,000 (MS), 0 (MS/MS); maximal number of peaks per spectrum, 100 (MS, MS/MS). No smoothing was applied and the peaks were not de-isotoped. To allow protein identification, the peak lists of the MS and MS/MS spectra were compared against theoretical mass data deduced from the proteins stored in the Nostoc 7120 data base of The Institute for Genomic Research (Release 7.0; 6128 protein entries). To this end, the search algorithm Mascot version 2.0 (Matrix Science, Boston, MA) was used in conjunction with the Mascot Daemon tool version 2.0. The search parameters were as follows: search mode, MS/MS ions search; enzyme, trypsin/P; protein mass, unrestricted; peptide ion mass tolerance, ± 30 ppm; fragment mass tolerance, ± 0.3 Da; maximum number of missed cleavage sites, 1; variable protein/peptide modifications, acetylation of N termini of proteins, modification of cysteines by acrylamide (propionamide), and oxidation of methionines. The primary identification criterion was a significant (p < 0.05) MASCOT search result with a total protein score ≥50. To increase the confidence in protein identification, the data base search results were manually validated, in particular MS/MS identifications based on few peptide assignments. For immunoblotting, proteins were electrophoretically transferred to nitrocellulose membranes. ClpC and NblA-GST fusion protein were detected by specific antisera. Antigen-antibody complexes were visualized with a goat anti-rabbit IgG-peroxidase conjugate and SuperSignal West Pico (Pierce) as chemiluminescent substrate. Construction of Plasmids for Expression of GST Fusion Proteins—The plasmid for expression of GST-tagged ClpC was constructed as follows: the chromosomal gene alr2999 (29Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Shimpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 205-213Crossref PubMed Scopus (576) Google Scholar) from Nostoc 7120 was PCR-amplified from total Nostoc 7120 DNA using primers clpC7 and clpC8, incorporating two BamHI sites for cloning. The PCR fragment was digested with BamHI and ligated into the expression vector pGEX-6P-1 (GE Healthcare) yielding plasmid pGEX-6P/ClpC. For expression of different NblA-GST fusion proteins, the nblA gene or its variants were amplified by PCR from plasmid pIC20RnblA and pIC20RnblA variants using primers 2.5 and 2.15, NdeI sites incorporated. The fragments were cloned into plasmid pGEX-2TK/NdeI to express NblA or NblA variants containing a C-terminal GST tag. The plasmid pGEX-2TK/NdeI was generated by mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using plasmid pGEX-2TK as template and the primers GEXN-For and GEXN-Rev. The α-subunits of phycocyanin (PC) and phycoerythrocyanin (PEC), respectively, were expressed N-terminal fused to a GST tag. For the construction of the expression plasmids, we amplified chromosomal genes cpcA (alr0529, α-subunit of PC) and pecA (alr0524, α-subunit of PEC) by PCR from total Nostoc 7120 DNA using primers pecGEX1 (BamHI site inserted) and pecGEX2 (EcoRI site inserted) or cpcGEX1 (BamHI site inserted) and cpcGEX2 (EcoRI site inserted), respectively. After digestion with BamHI and EcoRI, the PCR fragments were cloned into plasmid pGEX-2TK. All DNA constructs were confirmed by DNA sequencing. The constructs described in this paragraph are depicted in supplemental Fig. S1, C–G. Protein Overexpression, Purification, and in Vitro Binding Assays Using N-terminal GST-tagged NblA—Expression, purification, and site-directed mutagenesis of N-terminal GST-tagged NblA was performed as described earlier (25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). GST-NblA or its variants were bound to Glutathione-Sepharose 4B columns and incubated with soluble crude extracts from Nostoc 7120, prepared as described (17Baier K. Lehmann H. Stephan D.P. Lockau W. Microbiology. 2004; 150: 2739-2749Crossref PubMed Scopus (61) Google Scholar). After extensive washing, proteins bound to the resins were eluted with 20 mm glutathione in 50 mm Tris/HCl (pH 8.0) and analyzed by SDS-PAGE and immunoblotting. SDS-PAGE and immunoblots were performed as described above. PC subunits and GST were detected by specific antisera as described before (25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Protein Overexpression, Purification, and in Vitro Binding Assays Using C-terminal GST-tagged NblA or Its Variants—The C-terminal GST-tagged NblA or its variants were expressed in E. coli after induction with isopropyl 1-thio-β-d-galactopyranoside for 2–3 h at 30 °C. Fusion proteins were purified according to the manufacturer's protocol (GE Healthcare). The GST-ClpC fusion protein was expressed after induction with isopropyl 1-thio-β-d-galactopyranoside overnight at 18 °C. ClpC containing an N-terminal GST tag was bound to Glutathione-Sepharose 4B in sodium/potassium phosphate buffer (20 mm sodium/potassium phosphate buffer (pH 7.6), 200 mm KCl, 20 mm NaCl, 20 mm MgCl2, 10% glycerol, 5 mm ATP). After washing with buffer, ClpC was eluted by using PreScission™ Protease essentially as described by the manufacturer's directions for GST expression and purification systems (GE Healthcare). Protein concentrations were determined according to Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). NblA-GST was incubated with an equimolar concentration of ClpC (5 μm each) in sodium/potassium phosphate buffer (composition see above) for 2 h at 4 °C with ATP, AMPPNP, ATPγS, ADP, or GTP (each at 5 mm), and subsequently bound to Glutathione-Sepharose 4B overnight at 4 °C. After washing with the sodium/potassium phosphate buffer containing 0.5 mm ATP, AMPPNP, ATPγS, ADP, or GTP, proteins were eluted with 40 mm glutathione in 50 mm Tris/HCl (pH 8.0) containing 0.5 mm ATP, AMPPNP, ATPγS, ADP, or GTP, concentrated using deoxycholate/trichloroacetic acid precipitation (35Bensadoun A. Weinstein D. Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2722) Google Scholar), and analyzed by Tricine-Tris SDS-PAGE with 6 m urea as described (36Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). The in vitro binding assays using the different NblA-GST variants and ClpC were performed by the same procedure except that the incubation buffer contained 5 mm ATP and the washing buffer 0.5–1 mm ATP. The polyclonal anti-ClpC serum was purchased from Agrisera (Agrisera, Vännäs, Sweden, product number AS01 001). Protein Overexpression, Purification, and in Vitro Binding Assays Using GST-tagged PEC and PC—The N-terminal GST-tagged α-subunits of PC and PEC were expressed after induction with isopropyl 1-thio-β-d-galactopyranoside overnight at 18 °C and purified according to the manufacturer's protocol (GE Healthcare). The GST-ClpC fusion protein was expressed and purified as described above. NblA expression and purification was performed as described earlier (25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Protein concentrations were determined according to Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). GST-PEC or GST-PC was incubated with ClpC for 3 h at 4 °C in the presence or absence of NblA (each protein at 3 μm) in sodium/potassium phosphate buffer (composition see above) containing 5 mm ATP. Affinity purification of GST-PEC or GST-PC complexes using Glutathione-Sepharose was performed as described above with 0.5 mm ATP in the washing and elution buffers. The eluted proteins were analyzed by Tricine-Tris SDS-PAGE in the presence of 6 m urea as described (36Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). The Conserved N-terminal Region of NblA Is Relevant for in Vivo Function—We have previously shown that NblA specifically binds to the α-subunits of PC and PEC via amino acid residues in a short conserved motif at the end of the C-terminal helix (Fig. 1C and Ref. 25Bienert R. Baier K. Volkmer R. Lockau W. Heinemann U. J. Biol. Chem. 2006; 281: 5216-5223Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, the highest conserved stretch of amino acids among all known NblA proteins is located
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