Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

Abstract

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis . Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form . In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids . The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls . Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Phase-Specific Polypeptides and Poly(A)+ RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

“…The high-mol wt RNA was extracted as described by Schmidt et al (36). Eleven to 13 g of frozen cells were homogenized with 40 mL of 50 mM Tris-HCl (pH 9.0), 1% (w/v) SDS, and an equal volume of 90% (v/v) phenol in a Waring blender.…”

The light‐dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center in barley

“…The purification of RNA was achieved by phenol/chloroform extraction and isopropanol precipitation, essentially as described by Schmidt et al [34]. Following LiCl precipitation to remove tRNA and contaminating DNA [34], the RNA was washed twice with 70% ethanol and dissolved in water.…”

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

“…antibodies raised against polypeptide 11 react with all of the mature polypeptides and the two precursors (17). This feature presumably reflects the structural similarities in the mature polypeptides (18,21,23 Rather than using intact chloroplasts for processing ofpLHCP, we decided to use a fully in vitro system, obtaining the processing enzymes in the soluble fraction of the cell. This system allowed quantitative analyses of the conversion of the pLHCP to their…”

“…These proteins are synthesized on cytoplasmic ribosomes (9,11,16) as precursors 2000 to 4000 D larger than the mature forms (1,12,17,21,25). The precursors are taken up by the chloroplast and cleaved to the mature size.…”