The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit

Abstract

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA(C) protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA(C) does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA(C) to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA(C). Assays of epitope-tagged wild-type and mutant variants of CgtA(C) indicate that the C terminus of CgtA(C) is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA(C) alleles to function in vivo. Depletion of CgtA(C) led to perturbations in the polysome profile, raising the possibility that CgtA(C) is involved in ribosome assembly or stability.

FIG. 1.

CgtA_C cofractionates with the 50S ribosomal subunit by sucrose density centrifugation. CB15N cells were grown to logarithmic phase in PYE medium and harvested, and cell lysates were prepared and sedimented through a 15 to 45% sucrose gradient at 210,000 × g for 3 h (A) or a 20 to 40% sucrose gradient at 84,000 × g for 12 h (B). UV profiles were monitored at 280 nm. The positions of the 30S, 50S, and 70S monosome and polyribosome peaks relative to those of the fractions collected are indicated. Immunoblots of relevant fractions (10 μl of each 1-ml fraction) separated by SDS-12% PAGE and the levels of CgtA_C detected with anti-CgtA_C antibodies are shown below each polyribosome trace. S, 10 ng of purified CgtA_C; L, 1 μl of cell lysate (OD₂₆₀ of 0.1).

FIG. 2.

Salt dependence of the association of CgtA_C with free 50S ribosomal subunits. CB15N cell lysates (200 μl, OD₂₆₀ of 20) were sedimented through 15 to 45% sucrose gradients containing 0 to 200 mM NH₄Cl (as indicated) in 10-mM Tris-HCl (pH 7.5) buffer supplemented with either 10 mM MgCl₂ (A) or 10 mM MgSO₄ (B). Polyribosome profiles similar to that shown in Fig. 1 were obtained, and the fractions corresponding to the top (T) of the gradients and the 50S peaks (50S) were separated by SDS-12% PAGE. Each lane was loaded with 10 μl of a 1-ml fraction, and the levels of CgtA_C were detected by immunoblotting using anti-CgtA_C antibodies. S, 2 ng of purified CgtA_C.

FIG. 3.

The addition of excess guanine nucleotides does not affect the association of CgtA_C and free 50S ribosomal subunits. CB15N cell lysates (200 μl, OD₂₆₀ of 20) were preincubated on ice without (+None) or with 10 mM GDP (+GDP) or 10 mM GTP (+GTP) for 30 min prior to sedimentation through a 15 to 45% sucrose gradient supplemented with no nucleotide, 250 μM GDP, or 250 μM GTP, respectively. Polyribosome profiles similar to that shown in Fig. 1 were obtained, and the fractions corresponding to the top (T) of the gradients and the 50S peaks (50S) were separated by SDS-12% PAGE. Each lane was loaded with 10 μl of a 1-ml fraction, and the levels of CgtA_C were detected by immunoblotting using anti-CgtA_C antibodies. S, 2 ng of purified CgtA_C.

FIG. 4.

Gel filtration chromatography of a C. crescentus CB15N cell lysate reveals that the majority of CgtA_C elutes as a monomer or in a small complex. CB15N cell lysates (0.5 ml) were fractionated through a 100-ml (1.5 by 70 cm) Toyopearl HW-55S column (TosoHaas) at a flow rate of 0.4 ml/min in TMA buffer (10 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 100 mM NH₄Cl). (A) The elution of samples was monitored by UV absorbance at 280 nm, and 1-ml fractions were collected using elution volumes between 20 and 100 ml. The elution positions of control proteins are indicated. (B) The indicated fractions (10 μl each) were separated by SDS-12% PAGE. The levels of CgtA_C were detected by immunoblotting using anti-CgtA_C antibodies. S, 2 ng of CgtA.

FIG. 5.

Modification of the CgtA_C C terminus affects function. Serial dilutions of cultured JM1108 cells containing pMR20 plasmids expressing cgtA_C, no protein (None), cgtA_C-3HA, cgtA_C^1-347, or cgtA_C^1-347-3HA were spotted onto PYE+Xyl and PYE+Glu plates. The plates were incubated at 30°C for 2 days.

FIG. 6.

Growth of tagged CgtA_C GTP-binding domain mutants. Serial dilutions of cultured JM1108 cells containing pMR20 plasmids expressing cgtA_C, no protein (None), cgtA_C-3HA, cgtA_CT192A-3HA, or cgtA_CT193A-3HA were spotted onto PYE+Xyl and PYE+Glu plates. The plates were incubated at 30°C for 2 days.

FIG. 7.

The C-terminally 3HA-tagged CgtA_C proteins failed to cosediment with free 50S ribosomal subunits in the cell lysates. C. crescentus JM1108 cells expressing cgtA_C^1-347-3HA (second and third lanes), cgtA_C-3HA (fourth and fifth lanes), cgtA_C^T192A-3HA (sixth and seventh lanes), and cgtA_C^T193A-3HA (eighth and ninth lanes) episomally from pMR20 were grown to logarithmic phase in PYE+Xyl medium and harvested. Cell lysates were independently sedimented through a 15 to 45% sucrose gradient prepared in 10 mM Tris-HCl (pH 7.5)-10 mM MgCl₂-100 mM NH₄Cl. A total of 10 μl of each 1-ml fraction from the top of the gradient (T) and the free 50S ribosomal subunit peak (50S) was separated by SDS-12% PAGE. The results of immunoblotting with duplicate gels prepared using monoclonal anti-HA antibody (A) and anti-CgtA_C (B) antibodies are shown. S, 2 ng of purified CgtA_C.

FIG. 8.

Long-term depletion of CgtA_C reduces the levels of 70S ribosomes and polyribosomes. Wild-type C. crescentus cells (CB15N) and cells containing cgtA under the control of the Pxyl promoter (JM1108) were grown to mid-exponential phase in PYE+Xyl, washed with PYE, resuspended at low density in either PYE+Xyl or PYE+Glu medium, and grown at 30°C for 6 h. Cell lysates were sedimented through 15 to 45% sucrose gradients, and the resulting UV absorbance profiles were recorded.