Early events in the import/assembly pathway of an integral thylakoid protein

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Abstract
The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.