Translational defects in a mutant deficient in YajL, the bacterial homolog of the parkinsonism-associated protein DJ-1

Abstract

We report here that YajL is associated with ribosomes and interacts with many ribosomal proteins and that a yajL mutant of Escherichia coli displays decreased translation accuracy, as well as increased dissociation of 70S ribosomes into 50S and 30S subunits after oxidative stress.

FIG. 1.

Association of YajL with ribosomes. (A) Lysates from the wild-type strain were centrifuged for 150 min at 36,000 rpm in a SW41 rotor through 15 to 30% sucrose gradients in 10 mM Tris (pH 7.6), 10 mM magnesium acetate, and 50 mM ammonium chloride at 4°C. Ribosomes were detected by their absorbance at 260 nm and YajL by immunoblot analysis (fractions 5 to 29). Representative results are shown for three repeat experiments. (B) Sucrose gradient sedimentation profiles of extracts from the parental strain (filled circles) and from the yajL mutant (empty circles) after they were stressed with 1 mM hydrogen peroxide for 10 min. Ribosomal subunits were centrifuged for 4 h at 36,000 × g in the same conditions as described above. The distribution of YajL in fractions 17 to 30 from the parental strain was detected by immunoblot analysis.

FIG. 2.

Specific interaction between YajL and ribosomal proteins. Ribosomal proteins from the wild-type strain were prepared by incubating ribosomes (prepared as described in reference 9) overnight in 10 mM Tris (pH 7.6), 6 mM MgCl₂, 60 mM NH₄Cl, 1 mM 2-mercaptoethanol, 6 M urea, and 1 M LiCl at 0°C as described by Nomura and colleagues (18). The samples were centrifuged at 35,000 × g for 10 min in order to remove the precipitated RNA, and the ribosomal proteins (800 μg) were diluted 8-fold in 250 μl of 40 mM sodium phosphate (pH 8) and 100 mM NaCl in the absence (lane 1) or in the presence (lane 2) of 80 μg of purified His-tagged YajL. Proteins retained on nickel affinity columns (His-Select nickel affinity gel, 200-μl-bed-volume; Sigma-Aldrich) were analyzed by 15% acrylamide SDS-PAGE (silver stained) and identified by matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry (with an Applied Biosystems 4800 proteomics analyzer).

FIG. 3.

Effect of the yajL mutation on −1 and +1 frameshifting and on UAA and UAG readthroughs. (A) The wild-type (dark gray) and yajL mutant (light gray) strains were transformed with plasmids encoding β-galactosidase expressed with N-terminal frameshifts or nonsense codons or with the wild-type lacZ sequence (control). Bacteria were in the exponential phase of growth during the entire experiment. The β-galactosidase activities of the control strain and of the −1- and +1-frameshift strains were measured as described in reference . The values are averages of the results of at least three independent experiments. (B) The β-galactosidase activities of the control, frameshift, and nonsense strains were measured, and the values for β-galactosidase synthesis in the mutant strains relative to those for the parental strains are shown, after being normalized to the ratio obtained with the in-phase plasmid. The miscoding value of the parental strain was normalized to 1. (C) Complementation of the yajL mutant with its wild-type allele but not with the C106A allele. The in-phase and −1-frameshift-dependent β-galactosidase activities of the parental strain, the yajL mutant, and the yajL mutant complemented with plasmids pBAD33-yajL and pBAD33-yajLC106A were measured, and frameshift ratios calculated as described for panel B are shown for the mutant (white), the mutant complemented with the wild-type yajL gene (gray), and the mutant complemented with the C106A yajL gene (black). Bacteria were grown in LB medium containing 0.05% arabinose, 0.5 mM IPTG, and the appropriate antibiotics. (D) Measurement of the −1 frameshift as a function of the time elapsed after the onset of stress with 100 μM hydrogen peroxide. β-galactosidase activities of the wild-type strain and of the yajL mutant expressing either the in-phase β-galactosidase (wild type, light gray; mutant, white) or the −1-frameshift-dependent β-galactosidase (wild type, dark gray; mutant, black) are shown. Bacteria were in the exponential phase of growth and had similar doubling times during the entire experiment. The values are averages of the results of three independent experiments. (E) The −1 frameshift ratios between the yajL mutant and its parental strain were calculated from the results shown in Fig. 3D and described in the legend to Fig. 3B and are shown as functions of the time elapsed after the onset of stress with 100 μM hydrogen peroxide.