Localization of Carbamoylphosphate Synthetase and Aspartate Carbamoyltransferase in Chloroplasts

Abstract

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-rown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia okracca), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSnse, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed. The two enzymes, Gln-dependent CPSase' (EC 6.3.5.5) and ACTase (EC 2.1.3.2) are involved in the de novo biosynthesis of pyrimidine compounds in all organisms (see reviews 7, 17). In the first step of the pyrimidine pathway, CPSase catalyzes the synthesis of CarP from CO2 with Gln as the nitrogen donor and ATP as an energy source. The carbamoyl moiety of CarP is then transferred by ACTase to form CarAsp. Using 14C-labeled compounds , C02, CarP, and CarAsp are incorporated into UMP by intact cells ofexcised roots of Cucurbita pepo (10). Four enzymes, including CPSase and ACTase, of the de novo pathway were found in the cytosol fraction of Vinca rosea cells (9). These two results indicate the presence of the de novo pathway in cytosol of plant cells. Alternatively, plants and other organisms can convert the nucleosides and bases to nucleotides by the so-called salvage pathway. The UMP formed through either pathway is then phosphorylated to UTP, a part of which is used for the synthesis of CTP. Chloroplasts possess all the machinery required for protein synthesis, so, they are genetically autonomous in some ways. Genes in chloroplast DNA are transcribed by chloroplast RNA polymerases and the messages are translated on chloroplast ri-bosomes. Thus, intact isolated spinach chloroplasts are able to incorporate [3Hluridine into the RNA (6) and Acetobularia chloroplasts incorporate ['4C]uracil into RNA (18). These results indicate that uridine and uracil can be (a) transported across the envelope into chloroplasts, (b) converted to UMP via the salvage pathway, (c) phosphorylated to UTP, and (d) subsequently incorporated into RNA by RNA polymerase. On the other hand, ' Abbreviations: CPSase, carbamoylphosphate synthetase; ACTase, aspartate carbamoyltransferase: CarP, carbamoylphosphate; CarAsp, car-bamoylaspartate; OCTase, ornithine carbamoyltransferase (EC 2.1.3.3); ALAD, 5-aminolevulinic acid dehydratase (EC 4.2.1.24); PEPCase, phos-phoenolpyruvate carboxylase. it was reported (8) that isolated chloroplasts of Bryophyllum daigremontianum Berger incorporate the label from ['4C]orotic acid, an intermediate of the de novo pathway, into nucleotides and RNA in the presence of 5-phosphoribosyl-l-pyrophosphate and ATP. These results indicate that (e) orotate can be transported across the envelope and (f) chloroplasts possess some enzymes of the de novo pathway, those which convert orotate via orotidine monophosphate to UMP. It is not known whether chloroplasts contain the entire components required for pyrimidine de novo biosynthesis, or whether chloroplasts depend upon the cytosol for the synthesis of pyrim-idine precursors (orotate, uracil, or uridine). In this paper, we provide direct evidence for the presence of the first two enzymes of pyrimidine biosynthetic pathway, CPSase and ACTase, in chloroplasts purified from four plant species. The higher activity of ACTase relative to CPSase found in chloroplasts and reported in extracts of many organisms, is discussed in relation to the instability of CarP in situ and to the control of pyrimidine synthesis. MATERIALS AND METHODS Plant Materials. Radish seeds (Raphanus sativus var. Wase-Yonjunichi) were germinated and grown on moistened absorbent cotton in the dark at 22 to 25°C (19, 20). The dark-grown seedlings were exposed to continuous illumination 3 d after germination in a growth chamber (NK Biotron, LH-200 RD, 7.6 m W/cm2, 25C). Spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays) were grown in the field, and the leaves of 3-to 5-week-old plants were used for the isolation of chloroplasts. Extraction of Radish Cotyledons. For developmental studies, 50 to 100 cotyledon pairs were excised at selected time intervals, and homogenized in the presence of 10% (w/w) insoluble-PVP, with a mortar and pestle in 10 ml of chilled grinding medium containing 0.1 M Tricine-KOH (pH 7.8), 10 mm KC1, 3 mm Gln, 10 mM MgCl2, and 10 mm 2-mercaptoethanol. The homogenate was centrifuged at 20,000g for 20 min. A portion of the super-natant was passed through a column of Sephadex G-25, equili-brated with the grinding medium, and the eluate was used for enzyme assays (see below). Chl (19) and RNA (20) were estimated as described previously. Preparation and Purification of Chloroplasts. Chloroplasts were purified from greened radish cotyledons (3 d dark then 40 h light) or from spinach, soybean and corn leaves. Fifty g of cotyledons or leaves were homogenized with a blender in a solution containing 0.33 M sorbitol, 50 mm Tricine-KOH (pH 7.8), 2 mM EDTA, 1 mM MgCl2, 20 mM NaCl, 20 mM iso-ascorbic acid, 3 mM Gln, 4 mM DTT, and 0.2% BSA. After filtration through two layers of Miracloth, each filtrate was centrifuged at 170g for 3 min to remove cell debris. The crude chloroplast fraction obtained by centrifugation at 1,000g for 10 min was resuspended in the homogenizing medium. For sucrose 126