A regulated RNA binding protein also possesses aconitase activity.

Kaptain, Stamatina; Downey, William; Tang, Catherine; Philpott, Caroline C.; Haile, David J.; Orloff, David G.; Harford, Joe B.; Rouault, Tracey A.; Klausner, Richard D.

doi:10.1073/pnas.88.22.10109

Cited by 199 publications

(97 citation statements)

References 27 publications

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“…IRP-1 is homologous to, and shares 30% identity with, mitochondrial aconitase (11), an enzyme of the Krebs cycle, and in iron-replete cells IRP-1 also has aconitase activity (12,13). Importantly, 18 active site residues of mitochondrial aconitase are conserved in IRP-1, including the three cysteines (427, 503, and 506) (14,15) A second IRE-binding protein, IRP-2, has recently been characterized by gel retardation assays and then purified and cloned from a variety of mammalian tissues and cells (17)(18)(19)(20).…”

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confidence: 99%

Control of Transferrin Receptor Expression via Nitric Oxide-mediated Modulation of Iron-regulatory Protein 2

Kim

Ponka

1999

Journal of Biological Chemistry

107

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confidence: 99%

Control of Transferrin Receptor Expression via Nitric Oxide-mediated Modulation of Iron-regulatory Protein 2

Kim

Ponka

1999

Journal of Biological Chemistry

107

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“…In our accompanying study (37), we created a strain expressing the yeast mitochondrial aconitase Aco1, a protein that does not bind RNA (24). That strain had less catalytic activity in cell extracts than did the wild type and yet still had a late-stage sporulation delay, a defect very similar to that of the citB5 mutant strain (37).…”

Section: Discussionmentioning

confidence: 99%

“…subtilis aconitase is structurally related to eukaryotic IRP-1 (iron regulatory protein 1), also known as cytosolic aconitase (3). IRP-1 is an RNA binding protein that regulates, posttranscriptionally, the expression of iron receptor, utilization, and storage proteins (3,5,15,16,24). IRP-1 exists in two forms and acts as a sensor of iron concentration and oxidative stress.…”

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Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006

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Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of K -dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3 untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.Aconitase catalyzes the reversible isomerization of citrate and isocitrate in the second step of the tricarboxylic acid branch of the Krebs citric acid cycle. In Bacillus subtilis, the Krebs cycle is induced during late exponential phase in nutrient-exhausted medium and is responsible for the production of biosynthetic intermediates and ATP and for reducing power during stationary phase and sporulation. When induced, aconitase becomes one of the most abundant proteins in the cell (8). Induction of the aconitase gene, citB, is dependent on inactivation of two DNA binding proteins, CcpC (22) and CodY (25). Repression by CcpC is antagonized by citrate (22), whereas CodY loses repressing activity when the intracellular pools of GTP (32) and branched-chain amino acids (38) decrease. A citB null mutant is severely defective in spore formation and has a relative sporulation efficiency of 1.4 ϫ 10 Ϫ4 % compared to its wild-type parent (6). Previous work by Craig et al. (6) showed that the defect in sporulation of a citB null mutant is caused, in part, by the accumulation of citrate, a chelator of Mn 2ϩ and Fe 2ϩ . Chelation of divalent cations disrupts phosphorylation (activation) of Spo0A, the major transcription factor for early sporulation-specific genes (19). However, addition of excess Mn 2ϩ and Fe 2ϩ only partially overcomes the sporulation defect of an aconitase null mutant (6).When a constitutively active allele of spo0A is introduced into the citB null mutant, cells proceed past the stage 0 block in sporulation but are still blocked at a later stage (6). A similar result is seen when an aconitase null mutant is genetically modified to prevent synthesis of citrate (6); such cells proceed past stage 0 but do not complete sporulation efficiently. Taken together, these results suggest that citrate accumulation may be responsible for the stage 0 block, but aconitase may have an additional function unrelated to citrate metabolism that is important during the later stages of sporulation. The only other known activity of B. su...

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“…To do so, we expressed a heterologous aconitase, the Saccharomyces cerevisiae mitochondrial aconitase (Aco1), which is devoid of RNA binding activity (12,16,17), in a citB null mutant strain and tested the ability of the yeast enzyme to substitute for B. subtilis aconitase during growth and sporulation.…”

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Expression of Yeast Mitochondrial Aconitase in Bacillus subtilis

Serio

Sonenshein

2006

J Bacteriol

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Expression of yeast mitochondrial aconitase (Aco1) in a Bacillus subtilis aconitase null mutant restored aconitase activity and glutamate prototrophy but only partially restored sporulation. Late sporulation gene expression in the Aco1-expressing strain was delayed.Bacillus subtilis aconitase, encoded by the citB gene (6), is both an enzyme of the Krebs citric acid cycle and an RNA binding protein (1,6). The RNA binding function is similar to that of the eukaryotic cytoplasmic aconitase IRP-1 (iron regulatory protein 1) (2,4,8,9), but the physiological role of B. subtilis aconitase RNA binding is not known (1). A citB null mutant requires glutamate (Glt) for growth and is severely defective in sporulation, having a sporulation efficiency 10 6 -fold lower than that of the wild type (5). Part of the reason for this defect is intracellular and extracellular accumulation of citrate, which chelates divalent cations, reduces the pH, and prevents activation of the master transcriptional regulator of sporulation Spo0A, resulting in a blockage of sporulation at stage 0 (5). However, alleviation of these metabolic defects results in only partial restoration of sporulation (5,11,13,20), suggesting a nonenzymatic role for aconitase in sporulation, possibly through utilization of its RNA binding function.To assess the effect of the loss of aconitase RNA binding on sporulation, we sought to create a B. subtilis mutant strain that has aconitase enzyme activity but no RNA binding activity. To do so, we expressed a heterologous aconitase, the Saccharomyces cerevisiae mitochondrial aconitase (Aco1), which is devoid of RNA binding activity (12,16,17), in a citB null mutant strain and tested the ability of the yeast enzyme to substitute for B. subtilis aconitase during growth and sporulation.The coding sequence of Aco1 was cloned downstream of a B. subtilis promoter (Pspac*) and ribosome binding site, in plasmid pKM67 (13), and integrated into the chromosome of a citB null mutant, MAB160 [trpC2 pheA1 ⍀(citB::spc)] (5), at the nonessential amyE locus. Transformants were selected for glutamate prototrophy (Glt ϩ ) or for chloramphenicol resistance (Cam r ), a vector marker. Cam r transformants appeared during overnight incubation at 30°C, but most were Glt Ϫ . Glt ϩ transformants appeared only after 48 to 72 h at 30°C; the prolonged incubation period suggested that a mutation in the recipient cells might have been required to permit the appearance of the Glt ϩ transformants. Glt ϩ transformants grew in minimal medium at 30°C but not at 37°C. Note that laboratory strains of S. cerevisiae grow much better at 30°C than at 37°C (D. Dawson, personal communication).To investigate whether a mutation had occurred in the Glt ϩ transformants, chromosomal DNA from a Glt ϩ Cam r transformant was isolated and introduced again into MAB160. Transformants were selected either for the Glt ϩ or for the Cam r phenotype and were then tested for the unselected marker. All Glt ϩ transformants were Cam r , but only 5% of Cam r transformants were Glt ϩ . In...

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A regulated RNA binding protein also possesses aconitase activity.

Cited by 199 publications

References 27 publications

Control of Transferrin Receptor Expression via Nitric Oxide-mediated Modulation of Iron-regulatory Protein 2

Control of Transferrin Receptor Expression via Nitric Oxide-mediated Modulation of Iron-regulatory Protein 2

Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

Expression of Yeast Mitochondrial Aconitase in Bacillus subtilis

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