Abstract
Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.
Highlights
The experiments in the present communication use the light-harvesting chlorophyll a/b protein, LHCP,’ as a model precursor proteins, were incorporated into thylakoids to explore theprocess by which imported proteins are incorunder the described assay conditions
The precursor to the light-harvesting chlorophyll a/b protein (preLHCP) thawt as used in this and the following experiments was prepared by translation of hybrid-selected mRNA (3)
The results presented in this communication provide evidence foran activity in chloroplast lysates that can integrate preLHCP into thethylakoid bilayer
Summary
Vol 261, No 31, Issue of November 5, pp. 14804-14810, 19% Printed in U.S.A. Kenneth Cline Fruit Crops Department, University of Florida, Gainesuille, Florida 32612. The process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. Several other thylakoid precursor proteins in addition to preLHCP, butno stromal stromal compartment and thenbe incorporated into the thylakoid bilayer or lumen It ailsso aunique process in organelle biogenesis because the site of stable membrane integration is separatedfromthesite of membranetransport. The experiments in the present communication use the light-harvesting chlorophyll a/b protein, LHCP,’ as a model precursor proteins, were incorporated into thylakoids to explore theprocess by which imported proteins are incorunder the described assay conditions. This assumes a 100%recovery of membranes following the assay
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