Abstract
A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa α-subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- Pi :
-
phosphate
- IEP:
-
inner envelope membrane protein
- pIEP:
-
precursor form of IEP
- SSU:
-
small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase
- αIEP96pep :
-
peptide specific antiserum to IEP96
- αIEP96pol :
-
polyspecific antiserum to IEP96
References
Abad St, Clark E, Lamppa GK: Properties of a chloroplast enzyme that cleaves the chlorophyll a/b binding protein precursor. Optimization of an organelle-free reaction. Plant Physiol 90: 117–124 (1989).
Baldet P, Alban C, Axiotis S, Douce R: Localization of free and bound biotin in cells from green pea leaves. Arch Biochem Biophys 303: 67–73 (1993).
Block MA, Dorne A-J, Joyard J, Douce R: Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. J Biol Chem 258: 13273–13280 (1983).
Chua NH, Gillham NW: The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii. J Cell Biol 74: 441–452 (1977).
Cline K, Werner-Washburne M, Andrews J, Keegstra K: Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675–678 (1984).
deBoer AD, Weisbeek PJ: Chloroplast protein topogenesis: import, sorting and assembly. Biochim Biophys Acta 1071: 221–253 (1991).
Dreses-Werringloer U, Fischer K, Wachter E, Link TA, Flügge UI: cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Its transit peptide contains an amphiphilic α-helix as the only detectable structural element. Eur J Biochem 195: 361–368 (1991).
Flügge UI, Fischer K, Gross A, Sebald W, Lottspeich F, Eckerskorn C: The triose phosphate-3-phosphoglyceratephosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J 8: 39–46 (1989).
Flügge UI, Weber A, Fischer K, Lottspeich F, Eckerskorn C, Waegemann K, Soll J: The major chloroplast envelope polypeptide is the phosphate translocator and not the protein import receptor. Nature 353: 364–367 (1991).
Grossmann A, Bartlett S, Chua NH: Energy dependent uptake of cytoplasmically synthesized polypeptides by chloroplasts. Nature 285: 625–628 (1980).
Harlow E, Lane D: Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).
Hirsch St, Muckel E, Heemeyer F, vonHeijne G, Soll J: A receptor component of the chloroplast protein translocation machinery. Science 266: 1989–1992 (1994).
Huang L, Berkelman T, Franklin AE, Hoffman NE: Characterization of a gene encoding a Ca2+-ATPase-like protein in the plastid envelope. Proc Natl Acad Sci USA 90: 10066–10070 (1993).
Joyard J, Billecocq A, Barlett SG, Block MA, Chua N-H, Douce R: Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts. J Biol Chem 258: 10000–10006 (1983).
Joyard J, Block MA, Douce R: Molecular aspects of plastid envelope biochemistry. Eur J Biochem 199: 489–509 (1991).
Keegstra K, Youssif AE: Isolation and characterization of chloroplast envelope membranes. Meth Enzymol 118: 316–325 (1986).
Klein RR, Salvucci ME: Photoaffinity labeling a mature and precursor forms of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase after expression in Escherichia coli. Plant Physiol 98: 546–553 (1992).
Kondo H, Shiratsuchi K, Yoshimoto T, Masuda T, Kitazono A, Tsuru D, Anai M, Sekiguchi M, Tanabe T: Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. Proc Natl Acad Sci USA 88: 9730–9733 (1991).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Li H-M, Sullivan TD, Keegstra K: Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein. J Biol Chem 267: 18999–19004 (1992).
Li S-J, Cronan JE: The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 267: 16841–16847 (1992).
Nakajima H, Hirata A, Ogawa Y, Yonehara T, Yoda K, Yamasaki M: A cytoskeleton-related gene, US02, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol 113: 245–260 (1991).
O'Farrell PH: High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021 (1975).
Olsen LJ, Keegstra K: The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J Biol Chem 267: 433–439 (1992).
Salomon M, Fischer K, Flügge U-I, Soll J: Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide. Proc Natl Acad Sci USA 87: 5778–5782 (1990).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanger F, Nickler S, Coulson AR: DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci 74: 5463–5467 (1977).
Sasaki Y, Hakamada K, Suama Y, Nagano Y, Furusawa I, Matsuno R: Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant. J Biol Chem 268: 25118–25123 (1993).
Soll J: α-tocopherol and plastoquinone synthesis in chloroplast membranes. Meth Enzymol 148: 383–392 (1987).
Soll J, Waegemann K: A functionally active protein import complex from chloroplasts. Plant J 2: 253–256 (1992).
Soll J, Alefsen H: The protein import apparatus of chloroplasts. Physiol Plant 87: 433–440 (1993).
Theg StM, Scott SV: Protein import into chloroplasts. Trends Cell Biol 3: 186–190 (1993).
Towbin A, Staehlin 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).
vonHeijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).
Waegemann K, Paulsen H, Soll J: Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence. FEBS Lett 261: 89–92 (1990).
Waegemann K, Soll J: Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts. Plant J 1: 149–158 (1991).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hirsch, S., Soll, J. Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate. Plant Mol Biol 27, 1173–1181 (1995). https://doi.org/10.1007/BF00020890
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020890