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

Abstract

Abstract. 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) fluorescencemicroscopy 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.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria

“…Pea (Pisum sativum) chloroplasts were isolated from 10-d-old pea leaf and fractionated into outer and inner envelope membranes, stroma, and thylakoids as described previously (Waegemann et al, 1992).…”

Toc12, a Novel Subunit of the Intermembrane Space Preprotein Translocon of Chloroplasts

“…The degradation of p12 and Tic110 is not the result of postlysis digestion of inner envelope or stromal proteins, because the used amount of trypsin inhibitor effectively blocks the protease (lane 2) and Tic62 facing the stroma is not proteolysed (lane 3). In a second approach a low concentration of trypsin was added to right side out OEVs (Waegemann et al, 1992; Figure 2D, lanes 1-3) solubilized by Triton X-100 (unpublished data) or sonication (lanes 4 -6). As before, Toc34 was degraded even without solubilization (unpublished data).…”

The Chloroplast Protein Import Receptors Toc34 and Toc159 Are Phosphorylated by Distinct Protein Kinases

“…It is therefore reasonable to assume that the catalytic domain of OEK98 is also oriented in the similar manner. To test this assumption, outer envelope membranes, which are isolated as right-side-out vesicles (22) were incubated with increasing concentrations of trypsin and membrane fractions were separated from the supernatant by centrifugation. Activity of OEK98 was subsequently assayed by kinase renaturation in the presence of Toc34⌬TM.…”