Conformational requirements of a recombinant ferredoxin-NADP+ reductase precursor for efficient binding to and import into isolated chloroplasts.
The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
Ceccarelli
E A
EA
Molecular Biology Division, PROMUBIE, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Krapp
A R
AR
Serra
E C
EC
Carrillo
N
N
D001692:Biological Transport; D002736:Chloroplasts / Q000201:enzymology / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D004792:Enzyme Precursors; D005288:Ferredoxins; D011994:Recombinant Proteins; D009249:NADP; D014508:Urea