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

Abstract

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.

Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP, and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C

“…The studies described here confirm and extend results obtained during the study of in organello synthesized L subunit assembly into Rubisco (Barraclough and Ellis, 1980;Bloom et al, 1983;Roy, 1984, 1985;Cannon et al, 1986;Cannon, 1987;Chaudhari, 1988;Roy et al, 1988;Chaudhari and Roy, 1989), and, most recently, of L subunit assembly in chloroplast extracts (Hubbs and Roy, 1992). They also provide information that may help explain the notorious failure of higher plant L subunits to assemble into Rubisco when expressed in E. coli.…”

“…Recently, we described the in vitro synthesis and assembly of Rubisco L subunits (Hubbs and Roy, 1992). That work confirmed the identification of paused Rubisco L subunit translation intermediates (Mullet et al, 1986) and showed that incorporation of L subunits into Rubisco requires S subunits.…”

“…Unfortunately, the expression of genes for the higher plant Rubisco in Escherichia coli has not yielded an active enzyme (Gatenby, 1984;Gatenby et al, 1987). Previous studies have described assembly of higher plant Rubisco after protein synthesis in intact leaves , in intact chloroplasts (in organello) (Barraclough and Ellis, 1980;Bloom et al, 1983;Roy, 1984, 1985;Cannon et al, 1986;Cannon, 1987;Chaudhari, 1988;Roy et al, 1988;Chaudhari and Roy, 1989), and most recently in chloroplast extracts (in vitro) (Hubbs and Roy, 1992). Rubisco L subunits were the predominant product found in the chloroplast stroma after in organello or in vitro protein synthesis (Blair and Ellis, 1980;Milos and Roy, 1984;Hubbs and Roy, 1992).…”

“…Previous studies have described assembly of higher plant Rubisco after protein synthesis in intact leaves , in intact chloroplasts (in organello) (Barraclough and Ellis, 1980;Bloom et al, 1983;Roy, 1984, 1985;Cannon et al, 1986;Cannon, 1987;Chaudhari, 1988;Roy et al, 1988;Chaudhari and Roy, 1989), and most recently in chloroplast extracts (in vitro) (Hubbs and Roy, 1992). Rubisco L subunits were the predominant product found in the chloroplast stroma after in organello or in vitro protein synthesis (Blair and Ellis, 1980;Milos and Roy, 1984;Hubbs and Roy, 1992). Barraclough and Ellis (1980) found L subunits in association with a "large subunit binding protein" after synthesis in organello.…”

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

“…In the stroma a newly synthesized large subunit is found in association with cpn60, which is required for the light-dependent assembly of Rubisco (Roy, 1989;Gatenby and Ellis, 1990;Ellis and van der Vies, 1991;Hartman and Harpel, 1994). In vitro-synthesized large subunits can be assembled into functional Rubisco in the absence of thylakoid membranes as well (Hubbs and Roy, 1992). Rubisco holoenzyme has also been found loosely associated with thylakoids, both independently (Mori et al, 1984;Makino and Osmond, 1991) and as part of a functional multienzyme complex (Suss et al, 1993(Suss et al, , 1995.…”