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The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties

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Abstract

The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.

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Abbreviations

CNBr:

cyanogen bromide

IP:

isoelectric point

NCS:

N-chlorosuccinimide

NTA:

nitrilotriacetic acid

omp24:

outer envelope membrane protein of spinach chloroplasts

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SV8 protease:

Staphylococcus aureus V8 protease (Endoprotease Glu-C)

TPT:

chloroplast triose phosphate/phosphate translocator

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Authors and Affiliations

  1. Julius-von-Sachs-Institut für Biowissenschaften der Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082, Würzburg, Germany

    Karsten Fischer, Andreas Weber, Bettina Arbinger, Susanne Brink & Ulf-Ingo Flügge

  2. Max-Planck-Institut für Biochemie, Am Klopferspitz 18, D-82152, Martinsried, Germany

    Christoph Eckerskorn

Authors
  1. Karsten Fischer
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  2. Andreas Weber
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  3. Bettina Arbinger
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  4. Susanne Brink
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  5. Christoph Eckerskorn
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  6. Ulf-Ingo Flügge
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Fischer, K., Weber, A., Arbinger, B. et al. The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties. Plant Mol Biol 25, 167–177 (1994). https://doi.org/10.1007/BF00023235

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  • DOI: https://doi.org/10.1007/BF00023235

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