Protein Import into and Sorting inside the Chloroplast Are Independent Processes

Abstract

Plastocyanin is a nuclear-encoded chloroplast thylakoid lumen protein that is synthesized in the cytoplasm with a large N-terminal extension (66 amino acids). Transport of plastocyanin involves two steps: import across the chloroplast envelope into the stroma, followed by transfer across the thylakoid membrane into the lumen. During transport the N-terminal extension is removed in two parts by two different processing proteases. In this study we examined the functions of the two cleaved parts, C1 and C2, in the transport pathway of plastocyanin. The results show that C1 mediates import into the chloroplast. C1 is sufficient to direct chloroplast import of mutant proteins that lack C2. It is also sufficient to direct import of a nonplastid protein and can be replaced functionally by the transit peptide of an imported stromal protein. C2 is a prerequisite for intraorganellar routing but is not required for chloroplast import. Deletions in C2 result in accumulation of intermediates in the stroma or on the outside of the thylakoids. The fact that C1 is functionally equivalent to a stromal-targeting transit peptide shows that plastocyanin is imported into the chloroplast by way of the same mechanism as stromal proteins, and that import into and routing inside the chloroplasts are independent processes.

The chloroplast‐targeting domain of plastocyanin transit peptide can form a helical structure but does not have a high affinity for lipid bilayers

“…Thus, the transit peptide with a thylakoid-transfer domain may well be transported to lipid bilayers of the organelle surface, then the chloroplast-targeting domain forms a (partially) helical structure in the lipid environments. Supporting this interpretation, residues 1-43 of the plastocyanin transit peptide fused to mouse dihydrofolate reductase, which does not itself show a high affinity for lipid bilayers [14], could not be imported into chloroplasts; at least residues 1 -53 of the plastocyanin transit peptide were necessary to direct mouse dihydrofolate reductase into chloroplasts [7]. Plastocyanin with a deletion of residues 44 -64 of the transit peptide can still be imported, but in this case, the C-terminal hydrophobic residues in the thylakoid-transfer domain, Ah65 and Met66, remain intact and the following N-terminal region of the mature plastocyanin including Alal, Va13, Leu4, Leu5, etc., is also highly hydrophobic.…”

“…1) [6]. The detailed in vitro import studies, using deletion mutants of pPC, have revealed that a pPC derivative lacking residues 44 -64 of the transit peptide can be imported into isolated chloroplasts, while a deletion of residues 38 to 64 abolishes the ability of the pPC derivative to import plastocyanin but still leaves some ability to bind to the chloroplast surface [7]. It is also known that the processing of the pPC transit peptide takes place first around residue 40 in the stroma and after residue 66 (the end of the transit peptide) in the thylakoid lumen [6].…”

“…pFD, precursor of ferredoxin; pSS, precursor of the small subunit of ribulose-l,5-bisphosphate carboxylase; pPC, precursor of plastocyanin; PC(1-37) a chemically synthesized peptide corrcsponding the first 37 residues of the transit peptide of pPC; PC( 1 -43) a chemically synthesized peptide corresponding the first 43 residues of the transit peptide of pPC; p25, a chemically synthesized peptide corresponding to the presequence of yeast cytochrome-oxidase subunit IV; PamOleGroPCho, I-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PamOleGroPGro, 1-palmitoyl-2-oleoyl-sn-glycerophosphoglycerol ; acyl,GalGro, 1,2-diacyl[~-~-galactopyranosyl-(1 ' ---f 3)]-sn-glycerol; acylZGalZGro, 1,2-diacyl[a-D-galactopyranosy1-(1 4 6')P-~-galactopyranosyl(l + 3')J-sn-glycerol; lysoPtdCho, lysophosphatidylcholine. [7]. The C-terminal domain appears to function as a thylakoidtransfer domain and is cleaved off upon translocation across the thylakoid membrane by the second processing protease located in the thylakoid lumen or on the lumenal face of the thylakoid membrane [8].…”

The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems.

“…The transit signals, which are hydrophilic, basic and enriched in hydroxylated residues, are structurally and functionally equivalent to the presequences of imported stromal proteins (Ko and Cashmore, 1989;von Heijne et al, 1989;Hageman et al, 1990). In contrast, thylakoid transfer signals are strikingly similar in structural terms to signal sequences which direct the translocation of proteins across the endoplasmic reticulum and the bacterial plasma membrane (von Heijne et al, 1989).…”

Sec-dependent Thylakoid Protein Translocation

“…This complex import pathway can be broadly divided into two phases, the first of which involves the transport of a cytosolically synthesized precursor protein into the stroma, after which the stromal form is transported across the thylakoid membrane into the lumenal space (Hageman et al, 1986;James et al, 1989;Ko and Cashmore, 1989;Hageman et al, 1990). The two translocation events are directed by distinct signals in the presequences of lumenal proteins.…”