Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)+-glutamate dehydrogenase from the unicellular green alga Chiamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.

Section: Isolation and Purification Of Mitochondria From C Reinhardtiicontrasting

confidence: 50%

Section: Resultsmentioning

confidence: 60%

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

Moyano

Ramazanov²,

Cárdenas³

et al. 1992

“…They were then incubated at 4°C for 40 min. After adding 3 mL H20 the samples were centrifuged and the upper aqueous phase dried (10 (40). The PFP used in the assay of Fru-2,6-P2 was partially purified (235-fold) from 1 kg of potato (Solanum tuberosum) tubers, using a P-lI Cellulose Phosphate column (Whatman) and a Q-Sepharose (1 x 6.5 cm) column (Pharmacia) (44).…”

Section: Metabolite Extractionmentioning

confidence: 99%

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

Turpin¹,

Botha²,

Smith³

et al. 1990

The assimilation of NH4 causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoonolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH4 assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.Over the last decade there has been a significant increase in our knowledge of the control of carbon partitioning from starch to sucrose in photosynthetic tissue (34). In the proposed models, most of the carbon exported from the chloroplast is TP (see Table I for complete list of abbreviations). In the cytosol, Fru-2,6-P2 plays an important role in regulating the conversion of TP to sucrose through its effects on cytosolic It is well established that nitrogen exerts a major influence on respiratory carbon metabolism (1,9,15,16,31,(35)(36)(37)41). The assimilation of NH4' enhances starch breakdown and the flow of carbon into TCA cycle intermediates which are then used in amino acid biosynthesis. At present there is no model which provides an integrative picture of the mechanism by which NH4' assimilation regulates this carbon partitioning. In the present study we report changes in cellular metabolites during the dark assimilation of NH4' by the Nlimited green alga Selenastrum minutum. We also report preliminary data on the localization and regulation of PFK, PFP, and FBPase in this organism. Together, these results allow us to propose an integrative model for the control of carbon partitionin...

“…The localization of GDH in higher plant and algal chloroplasts has also been reported (7,19,27). In one study, the subunit-precursor of chloroplastic GDH was shown to be synthesized in the cytosol (19).…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study, GDH was found to be localized exclusively in the chloroplast of Chiamydomonas, and the enzyme was shown to be equally active with NAD and NADP (7). An ammonium inducible NADP-GDH was also reported to be located in the chloroplasts of Chlorella, however, its subunit-precursor was shown to be synthesized in the cytosol ( 19).…”

mentioning

confidence: 99%

A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

Bhadula

Shargool²

1991

The subcellular distribution of L-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and 35S-methionine. Using antibodies raised against purified GDH, we identified a 35S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.