Abstract
Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the ‘frxB gene’ identified from DNA sequence analysis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aebersold RH, Teplow DB, Hood LE, Kent SBH: Electroblotting onto activated glass: High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis. J Biol Chem 261: 4229–4238 (1986).
Bennoun P: Evidence for a respiratory chain in the chloroplast. Proc Natl Acad Sci USA 79: 4352–4356 (1982).
Chomyn A, Mariottini P, Cleeter MWJ, Ragan CI, Matsuno-Yagi A, Hatefi Y, Doolittle RF, Attardi G: Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase. Nature 314: 592–597 (1985).
Chua N-H, Bennoun P: Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: Wild-type and mutant strains deficient in photosystem II reaction center. Proc Natl Acad Sci USA 72: 2175–2179 (1975).
Dunn PP, Gray JD: Nucleotide sequence of the frxB gene in wheat chloroplast DNA. Nucl Acids Res 16: 348 (1988).
Garab, Gyözö, Lajkó F, Mustárdy L: Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration. Planta 179: 349–358 (1989).
Gfeller RP, Gibbs M: Fermentative metabolism of Chlamydomonas reinhardtii. II. Role of plastoquinone. Plant Physiol 77: 509–511 (1985).
Godde D: Evidence for a membrane bound NADH-plastoquinone-oxidoreductase in Chalmydomonas reinhardii CW-15. Arch Mirobiol 131: 197–202 (1982).
Hager DA, Burgess RR: Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzyme activity: Results with sigma subunit of E. coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem 109: 76–86 (1980).
Hsieh JC, Lin FP, Tam MF: Electroblotting onto glassfiber filter from an analytical isoelectrofocusing gel: A preparative method for isolating proteins for N-terminal microsequencing. Anal Biochem 170: 1–8 (1988).
Johnson DA, Gautch JW, Sportsman JR, Elder JH: Improved technique utilizing non-fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Technol 1: 3–8 (1984).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lemaire C, Wollman F-A, Bennoun P: Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: An example of mitochondria-dependent photosynthesis. Proc Natl Acad Sci USA 85: 1344–1348 (1988).
Matsubayashi T, Wakasugi T, Shinozaki K, Yamaguchi-Shinozaki K, Zaita N, Hidaka T, Meng BY, Ohta C, Tanaka M, Kato A, Maruyama T, Sugiura M: Six chloroplast genes (ndh A-F) homologous to human mitochondrial genes encoding components of the respiratory chain NADH dehydrogenase are actively expressed: Determination of the splice sites of ndh A and ndh B pre-mRNAs. Mol Gen Genet 210: 385–393 (1987).
Maione TE, Gibbs M: Association of the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardii with photoreduction and the oxyhydrogen reaction. Plant Physiol 80: 364–368 (1986).
Nie ZQ, Chang DY, Wu M: Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas. Mol Gen Genet 209: 265–269 (1987).
Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki W: Chloroplast gene organization deduced from compete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574 (1986).
Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Masubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohta C, Torazawa K, Meng BY, Sugita M, Deno H, Kampogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M: The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO J 5: 2043–2049 (1986).
Thompson JA: Isolation and characterization of DNA from different plastid types of Tropaeolum majus. Eur J Cell Biol 21: 37–42 (1980).
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
Wu M, Nie ZQ, Yang J: The 18-kDa protein that binds to the chloroplast DNA replicative origin is an ironsulfur protein related to a subunit of NADH dehydrogenase. Plant Cell 1: 551–557 (1989).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lin, CH., Wu, M. A ferredoxin-type iron-sulfur protein gene, frx B, is expressed in the chloroplasts of tobacco and spinach. Plant Mol Biol 15, 449–455 (1990). https://doi.org/10.1007/BF00019161
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00019161