A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes.
Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.
Guéra
A
A
Institut de Biologie Moleculaire des Plantes du CNRS, Strasbourg, France.
America
T
T
van Waas
M
M
Weisbeek
P J
PJ
D001667:Binding, Competitive; D001692:Biological Transport; D002451:Cell Compartmentation; D002736:Chloroplasts / Q000378:metabolism*; D007887:Fabaceae; D005288:Ferredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D007425:Intracellular Membranes / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D008954:Models, Biological; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010946:Plants, Medicinal; D011489:Protein Denaturation; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D005288:Ferredoxins; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate