Regulation of a glutamyl-tRNA synthetase by the heme status

Abstract

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the C(5) pathway. In this route Glu-tRNA is transformed to delta-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme.

Fig. 1.

In vivo aminoacylated or deacylated tRNAs. Total tRNA obtained from cells grown in Fe²⁺, S°, or Fe²⁺/ALA treated with periodate directly (−NaOH) or after deacylation (+NaOH) was electrophoresed on a polyacrylamide gel and transferred to a solid matrix. RNAs were hybridized to oligonucleotide probes specific for each tRNA analyzed. The positions of aminoacyl and deacylated tRNAs are indicated.

Fig. 2.

Effect of ALA in the endogenous activity of GluRS1. Cells were cultured in the presence of the indicated concentrations of ALA. Specific activity of GluRS1 in extracts from late exponentially growing cells was measured. The plot represents pmol of Glu-tRNA formed after 15 min (initial velocity). Lines on the top of the bars indicate values range.

Fig. 3.

Inmunodetection of GluTR and GluRSs. Whole exponentially growing cells cultured in Fe²⁺ or S⁰ (A) or Fe²⁺ supplemented with various concentrations of ALA (B) were electrophoresed under denaturating conditions, transferred to a solid matrix, and probed with antibodies against A. ferrooxidans GluRS1 (Top), GluRS2 (Middle), or GluTR (Bottom). Each lane was loaded with 1 μg of protein except for GluRS2 detection (from a separate experiment) where 20 μg was used.

Fig. 4.

Level of mRNAs encoding GluRSs and GluTR. Total mRNA was extracted from exponentially growing cells cultured in Fe²⁺, Fe²⁺/ALA, or S⁰ and subjected to RT-PCR using specific primers for GluRS1, GluRS2, or GluTR. CyD is the representative amplification of a loading control.

Fig. 5.

Inhibition of GluRS1 by hemin. (A) Relative activity of GST-GluRS1 (▴), rGluRS1 (●), and endogenous GluRS1 (■) treated with variable concentrations of hemin is shown. Initial velocities without added hemin measured as Glu-tRNA formed in 15 min were GST-GluRS1 (61.8 pmol/50 nM), rGluRS1 (46.8 pmol/50 nM), and endogenous GluRS1 (458 pmol/μg of total protein). (B) rGluRS1 was preincubated with hemin then incubated with 2 mM NADPH or NADP⁺, and the aminoacylation activity was measured after 15 min of incubation. Lines on the top of the bars indicate values range.