Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus

Abstract

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 Although all chlorophytes code for the LS and SS of this enzyme in the nucleus and chloroplast, respectively (11,30), in the chromophytic alga Olisthodiscus luteus, immunoprecipitation of protein products synthesized in the presence of the cytoplasmic ribosome inhibitor cycloheximide gives evidence that both subunits of RuBPCase are synthesized in the chloroplast (20). Expression of the LS and SS polypeptides in a linked transcription-translation system using a cloned 0. luteus ctDNA fragment demonstrates (20) definitively that both genes are encoded on the plastid genome. The rbcL and rbcS genes are present on a large 20 kb inverted repeat which also contains psbA and ribosomal RNA cistrons (20) (TP Delaney, RA Cattolico, unpublished data). To date, except in Chlamydomonas eugametos (10) and Pelargonium (17) where the rbcL gene is also present on an inverted repeat, the large subunit of RuBPCase is present in single copy on every chloroplast genome analyzed. Gene arrangement of rbcL and rbcS in the eukaryote 0. luteus appears to be similar to that observed in cyanelles (permanent blue-green algallike symbionts which are present in a diverse array of colorless host cells) and blue-green algae. The close proximity of rbcL and rbcS in 0. luteus suggests that both these genes may be under the control of a single promotor as seen in prokaryotes and cyanelles (16,25).It would be of interest to know how the chloroplast DNA

Analysis of Chromophytic and Rhodophytic Ribulose-1,5-Bisphosphate Carboxylase Indicates Extensive Structural and Functional Similarities among Evolutionarily Diverse Algae

“…The protocol developed to purify Rubisco from the unicellular alga Olisthodiscus luteus (28) was modified to isolate the enzyme from the filamentous red alga Griffithsia pacifica. Both Griffithsia and Olisthodiscus Rubisco holoenzymes sediment through a 10 to 40% linear sucrose gradient at approximately the same rate.…”

“…Standard carbon fixation and kinetic assays were performed as previously described (28 Protein concentrations were determined using the Amido Schwarz lOB dye binding assay (37). Equal amounts ofprotein were employed in all electrophoretic assays.…”

“…Olisthodiscus Rubisco enzyme was isolated and enzyme activity assayed as previously described (28). Briefly, 2 to 4 L of cells were disrupted with a French pressure apparatus in buffer A (25 mM Tris-HCl [pH 8.1], 10 mM MgCl2, 10 mM KC1, 1 mm DTT, 0.5 mM EGTA, 0.7 ug/mL leupeptin, 0.7 ,ug/mL pepstatin) and the homogenate subjected to 35% ammonium sulfate precipitation.…”

“…However, several functional parameters such as Km(CO2) (18,19,44), rate of activation in the presence of substrate RuBP (1,17), and the degree of competitive inhibition of carboxylation by 6-phosphogluconate (8,9,19) (28). Griffithsia pacifica (Kylin) was grown at 20°C in large culture dishes (10 cm diameter, 7.5 cm high) containing about 300 mL f/2 seawater (41) or in 2.8 L Fernbach flasks containing 1 L of medium.…”

Photoadaptation in marine phytoplankton: variations in ribulose 1,5-bisphos-phate activity

“…The genetics, structure and regulation of RuBPCase has been studied extensively in higher plants (e.g. Lorimer 1981, Yoeh et al 1981, Miziorko & Lorimer 1983, Akazawa et al 1984, Kobza & Seemann 1988, but not in microalgae (Estep et al 1978, Appleby et al 1980, Hobson et al 1985, Plumley et al 1986, Newman & Cattolico 1987. Despite the obvious biochemical and physiological importance of RuBPCase, there have been surprisingly few systematic studies on the relationships between RuBPCase, or the 8-carboxylating Li et al 1984, Smith & Platt 1985, Mortain-Bertrand e~ al.…”