New insights into the import mechanism of the ferredoxin precursor into chloroplasts

We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-02, Vol.267 (4), p.2548-2556
Main Authors: Pilon, M, de Kruijff, B, Weisbeek, P J
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
animal
animales
animals
Autoradiography
Biochemistry & Molecular Biology
Biological and medical sciences
Biological Transport
Cell physiology
chloroplaste
chloroplasts
Chloroplasts - metabolism
Chromatography, Gel
cisteina
citoplasma
cloroplasto
cysteine
cytoplasm
cytoplasme
Disulfides - metabolism
Dithiothreitol - metabolism
Electrophoresis, Polyacrylamide Gel
ferredoxin
Ferredoxins - metabolism
fisiologia vegetal
Fundamental and applied biological sciences. Psychology
Iodoacetamide - metabolism
Life Sciences & Biomedicine
Membrane and intracellular transports
metabolisme des proteines
metabolismo proteico
metalloproteine
metalloproteins
metalproteinas
Molecular and cellular biology
physiologie vegetale
pisum sativum
plant physiology
protein metabolism
Protein Precursors - metabolism
reduccion
reduction
Science & Technology
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Summary:We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in vitro, in a wheat germ lysate, synthesized preferredoxin is not stably associated with another protein. Import is dependent only on ATP and does not require the presence of cytosolic proteins. Translocation could be largely stimulated by the thiol reducing agent dithiothreitol (DTT). To determine whether DTT acts on the precursor or on the chloroplast, we modified the 5 cysteines in the precursor by a reaction with iodoacetamide, thereby preventing the formation of disulfide bridges in the precursor. The import of this modified precursor was still stimulated by the addition of DTT, indicating that DTT had a stimulating effect on the chloroplast import machinery. In the case of the modified precursor, the import must have taken place without iron-sulfur cluster attachment in the stroma. The modified precursor could be imported with a similar efficiency as the parent precursor showing that import takes place independently from cofactor assembly.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)45915-7