Isolation of Thylakoid Membrane Vesicles ofChlamydomonas reinhardiiChloroplasts That Are Able to Integrate and Importin VitroSynthesized Precursor Proteins

Abstract

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 anin vitrosystem using isolated intact thylakoid membrane vesicles was of major importance. Here we report the isolation of intact thylakoid membrane vesicles of the green algaeChlamydomonas reinhardiifor the generation of a similar algal system. Further we show successful transport of severalChlamydomonasprecursor 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 isolatedChlamydomonasthylakoid membranes.