Abstract
Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the DeltapH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the DeltapH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the DeltapH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the DeltapH-dependent pathway.
Highlights
Nuclear-encoded thylakoidal proteins are synthesized in the cytosol, are imported into the chloroplast stroma, and are subsequently inserted into or translocated across the thylakoid membranes
A fusion protein (23K-BCCP)1 consisting of a wheat 23-kDa protein of the oxygen evolving complex (23K) and Escherichia coli biotin carboxyl carrier protein (BCCP), which is efficiently biotinylated in vivo [25], was expressed in E. coli cells
Preparation of a Biotinylated Substrate, intermediate form of wheat 23K (i23K)-BCCP—In order to halt the translocation of substrate proteins for the ⌬pHdependent pathway across thylakoid membranes, we utilized a biotinylated 23K protein complexed with a stably folded and bulky avidin tetramer
Summary
Nuclear-encoded thylakoidal proteins are synthesized in the cytosol, are imported into the chloroplast stroma, and are subsequently inserted into or translocated across the thylakoid membranes. The Sec-dependent and the ⌬pH-dependent pathways appear to utilize some pathway-specific components, they could share common translocation pore in the thylakoid membranes. We tested whether the purified fusion protein can be imported into isolated thylakoids via the ⌬pH-dependent pathway like authentic i23K.
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