Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.

Abstract

Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations.' Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain.The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor. proteins of ferredoxin and plastocyanm are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are compete'nt to be assembled into holo forms.Most chloroplastic proteins are encoded in the nucleus and synthesized in the cytosol as higher molecular weight precursors with amino-terminal-extensions called transit peptides. They are imported into chloroplasts' posttranslationally. The import process is usually divided into the following steps: (i) binding of precursors to the outer envelope membrane; (ii) translocation of polypeptides across the outer and inner envelope membranes; (iii) removal of transit peptides by the stromal processing protease; and (iv) depending on the protein, either further sorting into other chloroplastic compartments and/or assembly into their respective complexes (1). Most import studies have been performed using an in vitro reconstituted system in which radiolabeled precursors are synthesized by in vitro transcription and translation from cloned genes and are subsequently imported into isolated chloroplasts.Precursors to ferredoxin (prFD) and plastocyanin (prPC) are two proteins that have been examined in transport studies (2). The mature forms of these proteins are metalloproteins that function in photosynthetic electron transport. One unsolved aspect of their biogenesis concerns assembly of their metal-ion prosthetic groups. Although these two proteins follow the general steps of import described above,' current evidence is inadequate to determine when during the import process prosthetic groups are added. An understanding of when the metal-ion centers are assembled will have important implications for possible import mechanisms. For example, if the metal-ion centers are added in the cytosol, the precursors would then have to carry the metal-ion centers across the envelope during import. If the metal-ion centers are assembled inside chloroplasts, this raises questions about whether assembly occurs before or after proteolytic processing. Imported precursors and mature-sized proteins may have different conformations and only one ofthem may be competent for prosthetic group assembly.Higher plant ferredoxin (FD) contains a 2Fe-2S-type ironsulfur center and functions exclusively in the stroma of chloroplasts. A pathway for introducing the iron-sulfur center into spinach FD in isolated chloroplasts has been described (3). Isolated chloroplasts were incubated with exogenous cysteine and the sulfur atoms ...

PAA1, a P-Type ATPase of Arabidopsis, Functions in Copper Transport in Chloroplasts

“…By contrast, holoplastocyanin was barely detectable in paa1-1 and paa1-3, and apoplastocyanin accumulated instead ( Figure 8A, untreated). Treatment with ascorbate and KCN, which converts holoplastocyanin to forms of apoplastocyanin with different electrophoretic mobilities on native gels (Li et al, 1990), indicated that the wild-type plants had much higher contents of plastocyanin polypeptides ( Figure 8A, ascorbate/KCN treated), a finding that was confirmed by denaturing SDS-PAGE ( Figure 8B). This difference was not apparent in the untreated samples separated on the native gel ( Figure 8A, untreated), presumably because the anti-plastocyanin antibody detected apoplastocyanin much more efficiently than holoplastocyanin.…”

“…might be explained by a lack of functional plastocyanin. To assess this possibility, thylakoid lumen proteins from wild-type and paa1 plants were subjected to native gel electrophoresis (Li et al, 1990) to separate holoplastocyanin (containing Cu) and apoplastocyanin (lacking Cu). In wild-type plants, plastocyanin was present predominantly in the holo form ( Figure 8A, untreated).…”

“…Because the chloroplast protein import machinery has a strong unfolding capacity (Guera et al, 1993;America et al, 1994), proteins such as plastocyanin are thought to acquire their cofactors after import into the organelle (for review, see Merchant and Dreyfuss, 1998). De novo cofactor insertion into plastocyanin was observed after in vitro translocation into the lumen (Li et al, 1990). The plastocyanin polypeptide is unstable in Chlamydomonas reinhardtii grown in Cu-deficient medium (Merchant and Bogorad, 1986;Li and Merchant, 1995), and under these conditions, a cytochrome c 6 is induced that functionally replaces plastocyanin.…”

In Vivo Competition between Plastocyanin and a Copper-Dependent Regulator of the Chlamydomonas reinhardtii Cytochrome c6 Gene

“…Because the copper-requiring step in the biosynthesis of plastocyanin (i.e. formation of a stable holoprotein) is compartmentalized in the lumen of the thylakoid membrane (15,18), we cannot definitively conclude that partitioning of copper between the regulator (in the cytosol or nucleus) and plastocyanin (in the lumen) is set by their own relative cellular stoichiometries and affinities for copper. Distribution of copper between the regulator and plastocyanin might, for example, be determined by the affinities and directionalities of intracellular copper-transporting systems.…”

Genome Analysis of Chlamydomonas reinhardtii Reveals The Existence of Multiple, Compartmentalized Iron–Sulfur Protein Assembly Machineries of Different Evolutionary Origins

“…The importance of Fe-S clusters for photosynthesis stimulated early studies in the assembly of these metal cofactors in chloroplasts using nuclear-encoded, imported ferredoxin as a model protein (Takahashi et al 1986;Li et al 1990). By adding 35 S-labeled cysteine to lysed spinach chloroplasts, it was shown that cysteine is the source of sulfur for the ½2Fe-2S cluster of ferre-doxin (Takahashi et al 1991b).…”