Conformational Requirements of a Recombinant Ferredoxin‐NADP⁺ Reductase Precursor for Efficient Binding to and Import into Isolated Chloroplasts

Abstract

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 threefold increase of the maximal import rates to 14000 molecules · min‐1· chloroplast‐1, with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 μM 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.

Precursors with Altered Affinity for Hsp70 in Their Transit Peptides Are Efficiently Imported into Chloroplasts

“…As shown in Table I, import of the wild type preFNR displayed K m and V max values of about 406 Ϯ 194 nM and 10,195 Ϯ 1,888 molecules of precursors imported per minute by plastid, respectively. These values are statistically significant and reasonably coincident with kinetics parameters previously obtained for this process (41)(42)(43). In each set of experiments, the wild type and the mutant precursors were analyzed in parallel using the same chloroplast preparation.…”

Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones

“…It has been reported that translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence [39]. Similarly, we have previously observed that the import efficiency of folded preFNR is increased by a factor of 30 when leaf extracts were present during import [58].…”

Phosphorylation Enhances Mitochondrial Targeting of GSTA4-4 through Increased Affinity for Binding to Cytoplasmic Hsp70

“…Interestingly, addition of 4 M urea to RRL-translated protein vastly increased the import competence (not shown). It is known that urea treatment and associated protein unfolding improve the import competence of some proteins (48,49). Based on this we postulate that reticulocyte Hsp70 either binds to GSTA4-4 inefficiently or is unable to properly unfold the nascent chains, thus contributing to inefficient import of this protein.…”