Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase / Oxygenase in Chloroplast Extracts 1

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits. Previous investigations have characterized the proteinsynthesizing apparatus of the chloroplast. By most criteria, chloroplast ribosomes resemble those of bacteria in both form and function (9, 10). Translation systems derived from Escherichia coli, rabbit reticulocyte, and wheat germ can express chloroplast transcripts (6, 7). Additionally, several laboratories have characterized translation systems derived from the chloroplast stroma, finding that they can express endogenous or heterologous genes or transcripts (2, 3, 7). Some of these systems have been reported to synthesize Rubisco large subunits (2, 6). Rubisco holoenzyme contains eight large subunits, synthesized in the chloroplast, and eight small subunits, synthesized ' This work was supported by a grant from the National Research Initiative Competitive Grants Program of the U.S. Department of Agriculture. The work reported here is taken from a thesis to be submitted by A.H. in fulfillment of the requirements for the Ph.D. degree at Rensselaer. in the cytosol (9, 28). Our laboratory has described the in vitro assembly into holoenzyme of large subunits synthesized in isolated intact chloroplasts ('in organello') (4, 22, 23). However, there have been no reports that in vitro-synthesized large subunits assemble into holoenzyme. Researchers interested in improving the catalytic activity of this enzyme have emphasized the agricultural importance of assembling the higher plant enzyme in an easily manipulated bacterial source (28). Cloned genes for higher plant Rubisco subunits have not yielded detectable Rubisco activity when expressed in E. coli (11, 13). Cloned Rubisco genes from cyanobacterial sources, on the other hand, did yield active Rubisco (12, 30). Considering the similar quaternary structure of the cyanobacterial and higher plant enzymes, the failure of the higher plant subunits to assemble in E. coli may be due to slight differences in the primary structures of the two proteins; alternatively, assembly of the higher plant enzyme may be dependent upon some factor (e.g., the higher plant chaperonin, Cpn6014) not available in the bacterial system (13), or the higher plant subunits may be sensitive to some conditions of the foreign synthesis environment. In any case, critical information leading to the assembly of this enzyme in bacterial systems may be gained from a study of its assembly in a native system. Therefore, we sought conditions of in vitro protein synthesis that would allow the subsequent assembly of the translation products into holoenzyme. We examined the available literature on chloroplastbased in vitro protein-synthesizing systems and chose to use extracts that can complete nascent peptides as a starting point. Previous experience with protein synthesis in intact chloroplasts suggested that the Rubisco large subunit would be the primary product of a runoff synthesis reaction. However, we were unable to observe the assembly of in vitro-synthesized large subunits into Rubisco until we optimized the system to suit our needs. Because we required easily detectable amounts of assembly-competent large subunits, we selected conditions that yielded the greatest total accumulation of radioactivity into protein, rather than the highest initial synthesis rate. Additionally, we monitored the SDS-PAGE profile of the translation products to optimize recovery of the completed Rubisco large subunit polypeptides. This report describes characteristics of the large subunit synthesis reaction as well as the small subunit-dependent assembly of the newly synthesized large subunits into Rubisco holoenzyme, under conditions