Crippling the essential GTPase Der causes dependence on ribosomal protein L9

Abstract

Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either the peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of Escherichia coli that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth der phenotypes, neither factor could rescue the GTPase activities in vitro. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised der mutant strain and show that the L9-dependent phenotype coincides with a cell division defect.

Fig 1

L9 on the ribosome. A rendering of a crystal structure of the E. coli ribosome with L9 conservation is shown in two views (Protein Data Bank files 2i2t and 2i2p). The 50S and 30S subunits are indicated with 23S rRNA in pink, and 16S rRNA is in slate. The locations of the peptidyltransferase center (PTC) and the P-site are highlighted. L9 is surface rendered in blue, with invariant amino acids in red. The hop-1 residue that affects translation bypass, Ser93, is colored green.

Fig 2

L9 improves the health of der mutants. A synthetic-lethality screen revealed mutants that grow better with an unstable reporter plasmid expressing L9. (A) Comparison of the parental screening strain to two recovered der mutants on an X-Gal indicator plate. The parental cells did not require L9 and turned white during colony development from plasmid loss. Cells that grew better with L9 maintained a blue color in the colony because plasmid-containing cells were more fit. The derT57I strain was sicker in the absence of L9 than the derE271K strain, evidenced by the relative colony sizes without the reporter plasmid. (B) Strains cured of the reporter plasmid were transduced to replace the rplI locus in the chromosome. A control transduction replaced the original ΔrplI::tet mutation with the ΔrplI::cat mutation and did not improve growth. Restoring rplI (rplI-cat) improved the health of both der mutants but not to the level of the parental cells with wild-type der. (C) Complementation of the ΔrplI derT57I mutant with a mock plasmid or plasmids encoding L9^1–149-FLAG-His₆ (full length), L9^1–53-FLAG-His₆ (N domain), or L9^65–149-FLAG-His₆ (C domain). The N domain alone complemented the small-colony phenotype as well as full-length L9. (D) Transformation with plasmids that express wild-type or mutant Der to test for trans-complementation of the chromosomal der alleles. Wild-type Der restored full health to each mutant (second column). Overexpression of either the T57I or E271K mutant improved the health of each mutant but did not sicken cells with wild-type der in the chromosome. Therefore, each der mutant is partially functional and recessive.

Fig 3

Locations of the L9-dependent Der mutants and T57I mutant suppressors. Shown is a rendering of Der from Thermotoga maritima (Protein Data Bank code 1MKY) showing the locations of T57 and E271 and the relative positions of four E. coli T57I mutant suppressors (A45V, R109G, T113A, and V158G, in green). The GDP bound in G domain 2 is pink. In this conformation of Der, the T57I mutation lies at the interface between G domain 1 and the KH domain. The E271K mutation is in switch II of G domain 2.

Fig 4

DerT57I and E271K are compromised in their GTPase activities. (A) Various concentrations of wild-type, T57I, and E271K Der proteins were evaluated in a regenerative GTPase assay using a GTP concentration that nearly saturated the wild type (1 mM). (Inset) Coomassie-stained SDS-PAGE of the purified Der proteins. Increasing the wild-type Der concentration increased the observed GTP hydrolysis at each concentration tested. The T57I and E271K mutants displayed a measureable hydrolysis rate above background only at high concentrations (∼2 μM for the T57I mutant and ∼1 μM for the E271K mutant). The error bars are the standard deviations from three measurements. (B) Michaelis-Menten kinetic analysis of each protein at various GTP concentrations (0.008 to 2 mM). The wild type was assayed at 0.5 μM and the T57I and E271K mutants were each assayed at 2 μM, and then the rates were converted to turnover rate per enzyme. The K_m and V_max values for the wild type were 0.22 ± 0.04 mM and 2.42 ± 0.16 min⁻¹, respectively, and these values for the E171K mutant were 0.25 ± 0.06 mM and 0.20 ± 0.01 min⁻¹, respectively. The rate of GTP hydrolysis by T57I was too low for fitting.

Fig 5

YihI complementation and stimulation of Der. YihI with a FLAG-His₆ tag on its C terminus was expressed from a plasmid and used for complementation studies and to overexpress the protein for purification. (A) L9 strains with wild-type der, derT57I, or derE271K alleles. YihI complementation was evaluated under noninducing conditions to reduce YihI toxicity. YihI expression partially complemented the derT57I and derE271K mutants but did not restore wild-type growth. (B) The stimulation of wild-type Der (0.5 μM) with and without YihI-FLAG-His₆ (5.0 μM) was measured with increasing KCl and is presented as fold stimulation. At high concentrations of potassium, YihI did not stimulate Der. (C) YihI was added to GTPase assays containing wild-type (0.5 μM) or mutant (2 μM) Der at a 10-fold molar excess in buffer containing 100 mM KCl. No significant stimulation of the T57I mutant and a slight activation of the E271K mutant were observed. (D) Under conditions that allowed approximately half-maximal YihI stimulation of 0.5 μM wild-type Der (YihI at 2.6 μM), GTPase activities were assayed in the presence of either the T57I or E271K mutant as a competitor (each at 5.0 μM). The observed activity of the wild type mixed with the T57I mutant was the sum of the stimulated wild type and the nonstimulated T57I mutant, indicating that T57I did not appreciably compete for YihI (arrows). The E271K mutant was partially stimulated by YihI, and the mixture displayed the sum of both stimulated rates.

Fig 6

Conditional L9 degradation reveals an unsuppressed derT57I phenotype. A ΔclpX derT57I strain supported with either L9-cont or L9-deg was transformed with a controllable ClpXP expression plasmid and maintained under noninducing conditions to reduce the accumulation of second-site suppressors. (A) Providing L9-deg to the derT57I strain greatly reduced the accumulation of second-site suppressors. On the left is a representative plate showing the presence of suppressed mutant contaminants when the ΔrplI derT57I strain was grown as an overnight culture without L9 support. On the right is a plate of rplI-deg derT57I ΔclpX cells containing pClpXP that were grown from an overnight culture to late exponential phase in glucose medium (ClpXP off) and then plated on arabinose to induce ClpXP and degrade L9-deg. All colonies were small and reminiscent of freshly isolated, unsuppressed ΔrplI derT57I strains. (B) Cultures of L9-cont and L9-deg were grown to exponential phase and then either treated with glucose (to repress ClpXP expression [circles and triangles]) or induced with arabinose (to express ClpXP [crosses and diamonds]). As each fast-growing culture neared the end of exponential phase, aliquots of each were diluted 10-fold into fresh medium to allow extended outgrowth. Separate aliquots were removed for Western analysis of the tagged L9 (top). L9-cont was stable and L9-deg was reduced to very low levels by the first sampling. The growth rate of the culture undergoing L9 degradation was reduced by 36% during the last outgrowth. (C) DIC micrographs of derT57I strains grown with L9 (L9-cont, pClpXP induced) or without L9 (L9-deg, pClpXP induced). Degradation of L9 caused the cells to become elongated. (D) The lengths of 100 cells from each of four different cultures grown with pClpXP induced for three outgrowths were measured from several micrographs and plotted along with their averages (long lines) and standard deviations. Average lengths (μm): L9-cont, der⁺, 2.53; L9-deg, der⁺, 2.53; L9-cont, derT57I, 2.84; L9-deg, derT57I, 4.65.