Isolation of thylakoid membrane vesicles of Chlamydomonas reinhardii chloroplasts that are able to integrate and import in vitro synthesized precursor proteins.
Once imported into the stroma, nuclear encoded proteins of the chloroplast have to be routed to their final compartment, e.g. the thylakoid membranes. Four different pathways have been reported for the translocation of precursor proteins across and for the integration of mature proteins into the thylakoid membranes in higher plants. To study the sorting of precursor proteins in chloroplasts of higher plants the generation of an in vitro system using isolated intact thylakoid membrane vesicles was of major importance. Here we report the isolation of intact thylakoid membrane vesicles of the green algae Chlamydomonas reinhardii for the generation of a similar algal system. Further we show successful transport of several Chlamydomonas precursor proteins into isolated thylakoids: Lumenal precursors were translocated into the vesicles resulting in the accumulation of their mature, thermolysin-insensitive forms and thylakoid membrane proteins were specifically integrated into isolated Chlamydomonas thylakoid membranes.
Rüfenacht
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Department of Chemistry and Biochemistry, University of Berne, Switzerland.
Boschetti
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D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013462:Sulfur Radioisotopes
D008565:Membrane Proteins; D010940:Plant Proteins; D011498:Protein Precursors; D013462:Sulfur Radioisotopes; D008715:Methionine