Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles

Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence

The precursor form of the major-harvesting chlorophyll a/b-binding protein (pLHCP) of chloroplast thylakoids was overproduced in E. coli cells and used to study the influence of soluble factors on post-translational protein import into isolated pea chloroplasts. pLHCP solubilised in 8 M urea was not import-competent. However, if pLHCP was dialysed in the presence of soluble proteins (leaf extract) after urea treatment, import competence was gained. Dialysis of pLHCP in the presence of leaf extract alters its protease sensitivity. Stremai proteins, ovalbumin, trypsin inhibitor or chloroplast lipids could not produce import competence of pLHCP. Two components from leaf extract seem to be necessary, one of which can be mimicked by purified hsc 70, the other one requiring ATP. We conclude that soluble proteins from outside the stromal compartment are necessary for post-translational import of proteins into chloroplasts

Transfer of a chloroplast-bound precursor protein into the translocation apparatus is impaired after phospholipase C treatment

We have studied the influence of phospholipase C treatment of intact purified chloroplast on the translocation of a plastid destined precursor protein. Under standard import conditions, i.e. in the light in the presence or 2 mM ATP translocation was completely abolished but binding was observed at slightly elevated levels. An experimental regime which allowed binding but not import of the precursor protein, i.e. in the dark in the presence of 10 μM ATP, demonstrated that translocation intermediates, normally detected at this stage, were missing in phospholipase treated chloroplasts. The precursor was completely sensitive to protease treatment, indicating that the transfer of the precursor from the receptor to the import apparatus was blocked by phospholipase treatment