Characterization of a Cytosolic Aconitase in Higher Plant Cells

Abstract

ABSTRACFProtoplasts obtained from sycamore (Acer pseadplUatw) cell suspensions were found to be highly intact. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured leaving the fragile amyloplasts lagely intact. Aconitase hydratase ( for 1 h resulted in a final yield of protoplasts near 100%. After digestion, the suspension was filtered through one layer of Miracloth (Krantex, Alfortville, France) that retained any undigested cell aggregates, and protoplasts were collected by centrifuging at 150g for 10 min and then washed twice with 50 ml of culture medium containing 0.5 M mannitol (suspension medium). Protoplasts were stored in suspension medium and were normally used within 2 to 3 h.Gentle Rupture of Protoplasts and Separation of the Organelles from the Cytosolic Fraction. Since sycamore cell protoplasts have an average diameter of 20 to 30 gm, a rapid and effective procedure for the gentle rupture of intact protoplasts (i.e. for stripping the cell membrane) is to pass protoplasts through a fine nylon mesh (20 ,um) affixed to the cut end of a 25-ml disposable syringe (20). Thus, protoplasts (equivalent to 20.106 cells) are pelleted after dilution with suspending medium and resuspended in about 5 ml of a basic medium: 0.5 M mannitol; 0.1% (w/v) BSA; 1% (w/v) insoluble polyvinylpyrrolidone (Kca 25, Serva); 5 mM DTT; 10 Mm leupeptin; 5 mm phosphate buffer (pH 7.4); these last parameters were chosen because they correspond to the phosphate concentration and the pH of the cytosolic compartment previously determined by 31P nuclear magnetic resonance studies (22). If the protoplasts are taken up and expelled through the 20 Mm nylon mesh two times, they will be completely ruptured. In order to separate the cytosolic fraction from the cell organelles, we subjected the broken protoplast fraction to centrifugation to yield a pellet largely free of cytosol and a supernatant enriched in cytosolic enzymes. In order to prevent starch grains from tearing through the plastid envelope, the centrifugal g force was gradually increased by using an automatic rate controller (Sorvall) and centrifugation was carried out in three steps (100g for 5 min, 500g for 5 min, and 12,000g for 10 min; Sorvall rotor SS34). Each supernatant was centrifuged in a new tube, and the three successive pellets were combined together. This procedure ruptures all the protoplasts leaving the fragile amyloplasts and mitochondria largely intact (17).Isolation of Amyloplastids. For the preparation of plastids, intact protoplasts resuspended in basic medium were passed through a fine nylon mesh as described above.

A Developmentally Regulated Aconitase Related to Iron-regulatory Protein-1 Is Localized in the Cytoplasm and in the Mitochondrion of Trypanosoma brucei

“…To our knowledge, this is the first direct evidence that an IRP-like aconitase functions in mitochondrial metabolism in a eukaryote. The dual subcellular localization resembles the situation in plant tissues, where 50 -90% of total aconitase activity or protein reside in the cytosol and function in the glyoxalate cycle (20,21,74,75). Plant aconitase purified from cytosol and mitochondria is indistinguishable with respect to kinetic parameters, molecular mass (90)(91)(92)(93)(94)(95)(96)(97)(98), and the EPR spectrum (20, 64, 74 -76).…”

The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

“…It is not the case in Saccharomyces cerevisiae, in which a unique gene encodes both mitochondrial and cytosolic ACOs [38,39]. In plants, several reports indicate that, as in yeast, both mitochondrial and cytosolic ACOs are encoded by the same genes, suggesting a dual targeting of the proteins [40][41][42]. Therefore the question we had to address was whether or not the three identified ACOs were targeted to the cytosol.…”

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

“…The subcellular localization of NADP-ICDH suggests that these forms may have different functions in plant metabolism. The cytosolic enzyme has been proposed to be involved in the supply of 2-oxoglutarate for chloroplastic glutamate synthase activity (Chen and Gadal, 1990b); in this pathway, citrate, either generated in the mitochondrion or stored in the vacuole, could be moved to the cytosol, where it could produce isocitrate via an active cytosolic aconitase (Brouquisse et al, 1987). Recently, this hypothesis has been supported (Hanning and Heldt, 1993) by measuring the flux through the tricarboxylic acid cycle in isolated spinach leaf mitochondria in the presence of metabolite concentrations similar to those occumng in the cytosol in vivo.…”