Liberation of zinc-containing L31 (RpmE) from ribosomes by its paralogous gene product, YtiA, in Bacillus subtilis

Abstract

We have found that alternative localization of two types of L31 ribosomal protein, RpmE and YtiA, is controlled by the intracellular concentration of zinc in Bacillus subtilis. The detailed mechanisms for the alternation of L31 proteins under zinc-deficient conditions were previously unknown. To obtain further information about this regulatory mechanism, we have studied the stability of RpmE in vivo and the binding affinity of these proteins to ribosomes in vitro, and we have found that liberation of RpmE from ribosomes is triggered by the expression of ytiA, which is induced by the derepression of Zur under zinc-deficient conditions.

FIG. 1.

(A) Growth characteristics of L31 mutants in CSM at 37°C. Symbols: ○, 168 (ytiA⁺ rpmE⁺); ▵, RIK802 (ytiA::erm); □, RIK803 (rpmE::spc); •, RIK814 (ytiA::erm rpmE::spc). (B and C) Effect of ytiA mutation (B) or overexpression of ytiA (C) on intracellular levels of RpmE. (B) Cells were incubated in CSM at 37°C with shaking and were collected at the indicated times. Crude extracts containing 60 μg of proteins were separated by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18), and transferred to polyvinylidene difluoride membranes (Millipore). Immunodetection procedures were carried out as described previously (13). An anti-RpmE antibody (12) and an anti-YtiA antibody (which was prepared from a rabbit immunized with purified YtiA protein) were used at a 1:1,000 dilution. (C) RIK812 cells carrying the inducible ytiA expression plasmid, pDGytiA, were grown in CSM at 37°C with or without 1 mM IPTG. Cells were collected at the indicated times after inoculation, and aliquots of extracts containing 60 μg of total proteins were used for Western blot analysis.

FIG. 2.

Effect of overexpression of YtiA on the relative stability of RpmE. RIK812 cells were grown in CSM at 37°C with or without 1 mM IPTG. Chloramphenicol (100 μg/ml) and rifampin (10 μg/ml) were added to the culture when the optical density reached ca. 0.2. Time zero was taken as being 5 min after the addition of the antibiotics, and cells were collected at the times indicated. Aliquots of extracts containing 50 μg of protein (○) (without IPTG) or 70 μg (•) (with IPTG) were used for Western blot analysis. Relative RpmE amounts were quantified with ImageQuant 5.2 software (Molecular Dynamics). Each result is the average of three determinations. The ordinate (expressed with a log scale) shows the percentage of RpmE remaining. Error bars, standard deviations.

FIG. 3.

RFHR 2D gel electrophoresis of ribosomes prepared from YtiA-overproducing cells. RIK812 cells were grown in CSM at 37°C with shaking with or without 1 mM IPTG and were harvested 2 h after inoculation. Crude ribosomes were then purified and analyzed by RFHR 2-dimensional electrophoresis, which was carried out as described previously (12). PhastGel Blue R (Amersham) was used to stain the gels.

FIG. 4.

(A, B, and C) Competitive incorporation of RpmE and YtiA into L31-depleted ribosomes. Crude ribosomes prepared from RIK814 (rpmE::spc ytiA::erm) cells (A) and purified RpmE and YtiA proteins were used for the incorporation assay as described in the text. (B and C) RFHR 2D gels of the ribosome fraction before (B) or after (C) centrifugation. (D, E, and F) Incorporation of YtiA into RpmE-containing ribosomes. Crude ribosomes obtained from RIK802 (ytiA::erm) cells (D) and purified YtiA protein were mixed. (E and F) RFHR 2D gels of the ribosome fraction before (E) or after (F) centrifugation.

FIG. 5.

Model of the replacement of RpmE by YtiA under zinc-deficient conditions. See the text for details.