Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.
When synthesized in Escherichia coli, the light-harvesting chlorophyll a/b-binding protein (LHCP) precursor accumulates in inclusion-like bodies (Abad, M. S., Oblong, J. E., and Lamppa, G. K. (1991) Plant Physiol. 96, 1220-1227). In this study we show that after solubilization in 6 M urea and dialysis into 20 mM Tris-HCl (pH 8.0) the recombinant LHCP precursor (preLHCP) was not found as a monomer (31 kDa), but instead produced a heterogeneous population of oligomeric complexes, ranging from 60-300 kDa as determined by gel filtration chromatography. Circular dichroism analysis indicated that the oligomers had folded structure, and that it was composed of both alpha-helix and beta-sheet. Approximately half of recombinant preLHCP found in these complexes was cleavable at the transit peptide-mature protein junction by a soluble chloroplast-processing enzyme in an organelle-free reaction. At 1.5 microM the recombinant precursor inhibited the import of radiolabeled preLHCP and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase generated by reticulocyte lysate translations. When chloroplasts were preincubated with the precursor, followed by their reisolation, import was still blocked, providing evidence that competition between recombinant preLHCP and these substrates occurred at the chloroplast per se. Recombinant preLHCP was visualized on the envelope by immunofluorescence microscopy, and its presence there was mediated by a thermolysin-sensitive factor.
Oblong
J E
JE
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Lamppa
G K
GK
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D015388:Organelles / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010946:Plants, Medicinal; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D014158:Transcription, Genetic
D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase