Synthesis and Turnover of the Chloroplast Coupling Factor 1 in Chlamydomonas reinhardi

Abstract

Studies on the biosynthesis of the chloroplast coupling factor 1 (CFI) in Cklamydemonas reixhardi have been initiated. The ratio of CF, to chlorophyll in the cell was shown to be independent of the density of the culture. No turnover of assembled CF1 could be detected, thus suggesting that CF1 was synthesized at a rate equivalent to that of net chlorophyll synthesis. A lag of between 5 to 7 minutes in the incorporation of radioactive precursor sulfate into assembled CF1 was measureable. This puts an upper limit on the pool size of any precursor to the assembled CF, complex. The pool size is estimated to be equivalent to 1% of the total CF1 in the cell.The CF,2 is a multisubunit (a, :, 'y, 6, e) enzyme complex responsible for the terminal events in photophosphorylation. The synthesis ofCF, is dependent upon both cytosolic and chloroplast ribosomes (see 10 for a review). The subcellular location of the synthesis of the polypeptides that comprise CF1 has been rigorously determined in several laboratories. Isolated, intact chloroplasts have been shown to be capable of synthesizing the a, 0, and e subunits of CF, (9,22,25). In addition, the synthesis of these subunits (both in vitro by intact chloroplasts, and in vivo) is sensitive to the antibiotic chloramphenicol, an inhibitor of 70 S type (i.e. chloroplast) ribosomes. The synthesis of the y and a subunits, however, is insensitive to chloramphenicol, but sensitive to cycloheximide, an inhibitor of cytosolic 80 S ribosomes (3,23,24). The use of a cell free translation system directed by nuclear encoded (i.e. poly A') RNA indicated that the 'y and 6 subunits are probably synthesized as precursors, with NH2-terminal leader sequences (25, 30). These two subunits are therefore presumed to be synthesized outside the chloroplast, and imported into the organelle posttranslationally (5). Recent studies have demonstrated that the subunits synthesized within the chloroplast are encoded for on chloroplast DNA (7, 8, 13-15, 18, 26, 30). So far, the genes for the cytosolically synthesized subunits have not been located, but are probably on nuclear DNA.The above described organization of the genes coding for a chloroplast enzyme complex requires that there be close cooperation and synchrony between the two cellular protein synthesizing systems, so that efficiency is maximized. nature of chloroplast DNA and the high concentration of ribosomes in the chloroplast suggest that the control of protein synthesis and assembly is likely to be quite complex.In Chlamydomonas reinhardi, the results of protein synthesis experiments in the presence of inhibitors specific for protein synthesis in one or the other compartment of the cell have indicated that CF1 subunits can be synthesized and assembled into the membrane-bound complex in the absence of synthesis of part of the complex (23; Fig. 1). This can occur only if a pool of unassembled CF, subunits exists within the cell, or if some of the CF1 subunits in the assembled complex are subject to degradation and replacement by newly sy...

Analysis of the Import of Carboxyl-Terminal Truncations of the 23-Kilodalton Subunit of the Oxygen-Evolving Complex Suggests That Its Structure Is an Important Determinant for Thylakoid Transport

“…This drastic reduction in lifetime is even more remarkable when one considers the minimal nature of the truncation; OE23-1A, for instance, is missing only 10 amino acids from its carboxyl terminus. Chloroplasts are known to contain proteases that quickly degrade subunits that are not immediately assembled (Bennett, 1981;Schmidt and Mishkind, 1983;Merchant and Selman, 1984), and some of these function in the thylakoid lumen (Merchant and Bogorad, 1986;Kuwabara and Suzuki, 1994;Li and Merchant, 1995). Although this appears to be a general mechanism for the regulation of subunit stoichiometry in protein complexes (Luzikov, 1986), the subunits of the OEC do not follow this pattern of assembly or degradation (Ettinger and Theg, 1991).…”

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

“…The constituent polypeptides of the thylakoid ATPase do not generally accumulate as unassembled proteins (Merchant and Selman, 1984). Consequently, their steady-state levels are determined by the synthesis, maturation, or as- Proteins from whole cells (WC) or chloroplasts (Cp) isolated as described in "Materials and Methods" were denatured in sample buffer and fractionated in a 15% SDSpolyacrylamide gel, transferred to nitrocellulose, and reacted with antiserum raised against BRP or Oee2, the 23-kD polypeptide of the oxygen-evolving complex, which is localized in the chloroplast lumen.…”

Nuclear Gene-Regulated Expression of Chloroplast Genes for Coupling Factor One in Maize

“…If CF1 of wild-type maize does not turn over significantly, as has been shown in Chlamydomonas (22), the observations that 12 d-old hcf*-38 seedlings contain 10% of normal levels of the a and ,8 subunits and that the a and # polypeptides are synthesized at about 60% of the wild-type rate, gives half-lives for the a and 3 subunits in hcjf*-38 of perhaps 12 h.…”