Abstract
Abstract Chloroplast disc membranes were prepared from Chlamydomonas reinhardi y-1 after etiolated cells were exposed to light and allowed to produce chlorophyll and disc membranes for 8 to 8½ hours in liquid culture. Most, and possibly all, of the disc membrane proteins were synthesized during this period of membrane formation. The following results showed that both chloroplast and cytoplasmic ribosomes were involved in synthesizing these proteins. (a) Inhibition by chloramphenicol of the synthesis of polypeptides by chloroplast ribosomes did not prevent formation of the membrane structure in the cells. These membranes therefore contained primarily polypeptides synthesized in the cytoplasm, which became labeled when 14C-arginine was added to the cell culture along with chloramphenicol. (b) After 8½ hours in the presence of chloramphenicol the cells were transferred to medium containing cycloheximide, which inhibited protein synthesis by cytoplasmic ribosomes and prevented further membrane formation. During the following 2 hours in the light, the existing membranes became labeled when 3H-arginine was added to the culture medium, indicating that polypeptides were synthesized by chloroplast ribosomes and were incorporated into the membranes. The site of synthesis of individual membrane polypeptides was identified in such experiments by the distribution of 14C and 3H in fractions separated by polyacrylamide gel electrophoresis. Several membrane polypeptides were synthesized apparently only in the chloroplast, whereas most were synthesized in the cytoplasm. Among those synthesized in the cytoplasm were two fractions that together accounted for about 36% of the total protein stain on gels containing membrane proteins from control cells but about 43% of the total protein stain on gels containing membrane proteins from cells treated sequentially with chloramphenicol and cycloheximide. About one-half of the labeled arginine incorporated into protein in the cytoplasm was contained in these two fractions. These fractions were estimated to have molecular weights of 2.1 and 2.4 x 104. During the incubation of the cells in the presence of cycloheximide the density of the membranes decreased from 1.18 g per cm3 to 1.15 to 1.16 g per cm3, apparently as the result of the continued incorporation of lipids into existing membranes under these conditions.
Highlights
MethodsIsolation of Chloroplast Membranes-C. reinhardi y-l was grown as described previously (1, 3)
Of the synthesis of polypeptides by chloroplast ribosomes did not prevent formation of the membrane structure in the cells
Chloroplast disc membranes were prepared from Chlamydomonas reinhardi y-l after etiolated cells were exposed to light and allowed to produce chlorophyll and disc membranes for 8 to 8% hours in liquid culture
Summary
Isolation of Chloroplast Membranes-C. reinhardi y-l was grown as described previously (1, 3). Yellow cells were obtained after growth in the dark at 25” for 4 days and suspended to a density of 6 X lo cells per ml of medium supplemented with KH2P04 (3). 500-ml Erlenmeyer flasks, mounted on a rotating platform, and exposed to light from white fluorescent lamps at an intensity of Chloroplast Membrane Polypeptides Vol 245, No 17. 1 mM MgC12, suspended in the same solution to a density of about 2.5 X 1Os cells per ml, and passed once through a chilled. A solution of 0.1 M EDTA, pH 7.2, was added to provide a final concentration of 3 mM, and 10 ml of the sample were layered on a discontinuous gradient consisting of 20 ml of 0.5 M sucrose containing 1 mM EDTA, pH
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