Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein translocation across the thylakoid membrane--a tale of two mechanisms.

In vitro reconstitution assays have been used in recent years to probe the mechanisms by which a variety of cytosolically synthesised proteins are transported across the thylakoid membrane within higher plant chloroplasts. The emerging data suggest that two distinct mechanisms operate. Translocation of a subset of lumenal proteins, namely the 23 kDa and 16 kDa proteins of the oxygen-evolving complex, and of the CFo2 protein (an integral membrane protein), requires only the presence of the thylakoidal delta pH. In contrast, two other lumenal proteins, the 33 kDa oxygen-evolving complex protein and plastocyanin, require also the presence of ATP and at least one stromal factor for efficient transport into isolated thylakoids to take place.

Protein targeting across the three membranes of the Euglena chloroplast envelope.

A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes. Preparation of functional chloroplasts with intact envelope membranes has been optimized. Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62%. Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors. The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa). A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported. Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope. Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size. However, this was not imported into Euglena chloroplasts, although again it bound to them. These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence. The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.

Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol and targeted into the thylakoid lumen by bipartite presequences. In this report, we describe conditions for the efficient import of each of these proteins by isolated pea thylakoids. Import of the 33-kDa protein requires both light and stromal extract. The probable function of the stromal extract is to provide stromal processing peptidase to remove the first "envelope transit" signal of the presequence. Import of the 23-kDa protein is also driven by light, but stromal extract is not required for import; furthermore, efficient import is still observed if the precursor is modified to completely block cleavage by residual stromal processing peptidase activity. The intermediate form of the 23-kDa protein, which is generated by incubation of the precursor protein with stromal processing peptidase, is also efficiently imported. The results indicate that the thylakoidal protein transport system can import both the precursor and intermediate forms of the 23-kDa protein, but probably only the intermediate form of the 33-kDa protein.

Transport of proteins into chloroplasts. The thylakoidal processing peptidase is a signal-type peptidase with stringent substrate requirements at the -3 and -1 positions.

The transport of proteins across the thylakoid membrane in higher plant chloroplasts is usually mediated by an amino-terminal peptide extension which is subsequently removed by a specific thylakoidal processing peptidase. We have previously shown that the reaction specificity of this enzyme is very similar to those of signal peptidases located in the endoplasmic reticulum and bacterial plasma membrane. In the present report, the reaction mechanism of the thylakoidal peptidase has been investigated by substituting a variety of amino acids for the alanine residues at the -3 and -1 positions of a thylakoid lumen protein precursor. Small neutral side chains are known to be essential at these positions for cleavage by signal peptidases, and we find that these residues likewise play a critical role in defining the thylakoidal processing peptidase cleavage site. However, the requirements of the thylakoidal enzyme at these sites are significantly more restrictive than those of the bacterial or endoplasmic reticulum peptidases. Whereas leucine at the -3 position in the substrate is tolerated by the latter two enzymes, cleavage by the thylakoidal peptidase is almost completely inhibited. At the -1 position the presence of alanine appears to be critical; substitution of this residue by glycine, serine, threonine, leucine, lysine, or glutamate leads to either substantial or complete inhibition of cleavage at this site. Substitutions at either -3 or -1 which blocked cleavage at the correct site led to cleavage taking place at an alternative site, probably after the -21 residue.

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles.

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.