No abstract

Plastocyanin, a photosynthetic electron carrier functional in the chloroplast lumen, is synthesized in the cytosol as a precursor (preplastocyanin) with an amino-terminal transit sequence. This transit peptide contains the information specifying import into and routing within the chloroplasts and is removed in at least two steps. An intermediate is observed in the stroma after the transport of preplastocyanin through the chloroplast envelope; mature plastocyanin is present in the lumen, after transport over the thylakoid membrane. We show here that the stromal processing protease is not responsible for both processing events. It cleaves the precursor protein only to the intermediate size and a novel protease located in the thylakoids processes this intermediate to the mature protein. This second protease recognizes the processing intermediate but not the precursor. Thus plastocyanin import involves cleavage at the intermediate processing site mediated by the stromal protease and then cleavage at the mature processing site mediated by the thylakoid protease.

Protein import into chloroplasts requires the movement of a precursor protein across the envelope membranes. The conformation of a precursor as it passes from the aqueous medium across the hydrophobic membranes is not known in detail. To address this problem we examined precursor conformation during translocation using the chimeric precursor PCDHFR, which contains the plastocyanin (PC) transit peptide in front of mouse cytosolic dihydrofolate reductase (DHFR). The chimeric protein is targeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analogue of the substrate of DHFR. Methotrexate strongly inhibits DHFR import into yeast mitochondria (M. Eilers and G. Schatz, Nature 322 (1986) 228-232), presumably because the precursor must unfold to cross the membrane and it cannot do so when complexed with methotrexate. We show here that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degradation once inside chloroplasts. The processed protein is localized in the stroma, indicating that import into thylakoids is impeded. Protease sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation across the envelope.

No 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.