In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum: the transferase activity of R. rubrum GlnD is regulated by alpha-ketoglutarate and divalent cations but not by glutamine

Abstract

P(II) proteins have been shown to be key players in the regulation of nitrogen fixation and ammonia assimilation in bacteria. The mode by which these proteins act as signals is by being in either a form modified by UMP or the unmodified form. The modification, as well as demodification, is catalyzed by a bifunctional enzyme encoded by the glnD gene. The regulation of this enzyme is thus of central importance. In Rhodospirillum rubrum, three P(II) paralogs have been identified. In this study, we have used purified GlnD and P(II) proteins from R. rubrum, and we show that for the uridylylation activity of R. rubrum GlnD, alpha-ketoglutarate is the main signal, whereas glutamine has no effect. This is in contrast to, e.g., the Escherichia coli system. Furthermore, we show that all three P(II) proteins are uridylylated, although the efficiency is dependent on the cation present. This difference may be of importance in understanding the effects of the P(II) proteins on the different target enzymes. Furthermore, we show that the deuridylylation reaction is greatly stimulated by glutamine and that Mn(2+) is required.

FIG. 1.

Uridylylation of R. rubrum GlnB. GlnB (0.5 μM) was incubated in a uridylylation reaction mixture with 0.13 μM R. rubrum GlnD, 3 mM MnCl₂, 60 μM α-ketoglutarate, 2 mM ATP, and 0.5 mM UTP, supplemented with [α-³²P]UTP. Samples were withdrawn from the reaction mixtures after 0, 2, 5, 10, and 20 min of incubation and stopped by addition of SDS cocktail. The samples were subjected to SDS-PAGE and visualized by autoradiography in panel A or Western blotting in panel B, immunoblotted with antibodies against R. rubrum GlnB. In panel B, the lower band corresponds to unmodified GlnB and the upper to the modified form of GlnB.

FIG. 2.

Effect of MnCl₂, ATP, and α-ketoglutarate (α-KG) on uridylylation. (A) Uridylylation of GlnB (0.5 μM) with 0, 0.3, 0.6, 3.0, 6.0, or 16 mM MnCl₂ together with 2 mM ATP, 60 μM α-ketoglutarate, 0.13 μM GlnD, and 0.5 mM UTP supplemented with [α-³²P]UTP. (B) Effect of the absence of ATP or α-ketoglutarate on uridylylation of GlnJ (0.5 μM). The assays also contained 3 mM MnCl₂, 0.13 μM GlnD, and 0.5 mM UTP supplemented with [α-³²P]UTP. Uridylylation was assayed by incorporation of [α-³²P]UMP as described in Materials and Methods. Samples were withdrawn from the reaction mixtures after 20 min of incubation and stopped by addition of SDS cocktail.

FIG. 3.

The effect of α-ketoglutarate on uridylylation of GlnB with either MgCl₂ or MnCl₂. The assay contained 0.5 μM GlnB, 2 mM ATP, 0.13 μM GlnD, and 0.5 mM UTP supplemented with [α-³²P]UTP. Twenty-five millimolar MgCl₂ (▪) or 3 mM MnCl₂ (•). Samples were withdrawn from the reaction mixtures after 20 min of incubation and stopped by addition of SDS cocktail. The incorporation of [α-³²P]UMP at 300 μM α-ketoglutarate was set to 100% for both MgCl₂ and MnCl₂. No more labeling was detected at α-ketoglutarate concentrations higher than 300 μM. The samples were subjected to SDS-PAGE and visualized by autoradiography. The amount of labeled, uridylylated GlnB was quantified using the Image Quant program. The data shown are representative of at least three independent experiments for both MgCl₂ and MnCl₂.

FIG. 4.

Effect on uridylylation of Mg²⁺ or Mn²⁺. The three P_II paralogs (0.5 μM) were incubated with either 250 μM α-ketoglutarate and 25 mM Mg²⁺ (lanes 1, 3, and 5) or 60 μM α-ketoglutarate and 3 mM Mn²⁺ (lanes 2, 4, and 6). For both divalent cations used, 2 mM ATP, 0.13 μM GlnD, and 0.5 mM UTP, supplemented with [α-³²P]UTP, were included in the reaction mixture. Samples were withdrawn from the reaction mixtures after 20 min of incubation and stopped by addition of SDS cocktail. (A) Autoradiogram showing incorporation of [α-³²P]UMP into GlnB (lanes 1 and 2), GlnJ (lanes 3 and 4), or GlnK (lanes 5 and 6). (B) Coomassie-stained SDS-PAGE showing the levels of P_II proteins loaded in panel A. (C) Histogram showing the difference in incorporation of [α-³²P]UMP between the R. rubrum P_II proteins with Mg²⁺ (dark) or Mn²⁺ (gray) in the uridylylation reaction. The amount of labeled, uridylylated GlnB, GlnJ, or GlnK was quantified using the Image Quant program. The data shown are from at least three independent experiments. UTase, uridylyltransferase.

FIG. 5.

The effect of glutamine on uridylylation. In panels A, B, and C, 3 mM MnCl₂, 2 mM ATP, 60 μM α-ketoglutarate, and 0.5 mM UTP supplemented with [α-³²P]UTP were included in the reaction mixture. (A) GlnB (0.5 μM) with 0.13 μM purified R. rubrum GlnD. (B) GlnB (15 μM) with 50 μg E. coli GlnD activity. (C) Fifteen micromolar GlnB with R. rubrum N+ extract (120 μg protein). Lane 1, no glutamine added; lane 2, 10 mM glutamine added. Samples were withdrawn after 0 or 20 min of incubation and stopped by addition of SDS cocktail.

FIG. 6.

Deuridylylation activity of purified R. rubrum GlnD. The reaction mixture contained 0.4 μM GlnB-UMP, 1.3 μM GlnD, 3 mM MnCl₂, 500 μM α-ketoglutarate, 2 mM ATP, and 10 mM glutamine. The activities were estimated as the decrease in labeled [α-³²P]UMP-GlnB. Samples were withdrawn from the reaction mixtures after 0, 10, or 40 min and stopped by addition of SDS cocktail. The samples were subjected to SDS-PAGE and visualized by autoradiography (A) or Western blotting (B), immunoblotted with antibodies against R. rubrum GlnB.

FIG. 7.

Stimulation of the deuridylylation activity of purified R. rubrum GlnD. The data are from at least three independent experiments showing percentages of [α-³²P]UMP removed after 40 min of incubation for both GlnB-UMP (dark) and GlnJ-UMP (gray). For every pair of columns, 0.4 μM GlnB-UMP/GlnJ-UMP, 1.3 μM GlnD, and 3 mM MnCl₂ were added to the deuridylylation mixture. The first pair of columns shows deuridylylation with 500 μM α-ketoglutarate, 2 mM ATP, and 10 mM glutamine. The second pair shows deuridylylation without 2 mM ATP, and the third without 500 μM α-ketoglutarate. The last pair of columns shows deuridylylation without 10 mM glutamine. The data are from at least three independent experiments showing percent [α-³²P]UMP removed after 40 min of incubation for both GlnB-UMP and GlnJ-UMP.

FIG. 8.

Effect of glutamine on deuridylylation activity of purified R. rubrum GlnD. The uridylyl-removing activity was estimated as a decrease in labeled [α-³²P]UMP-GlnJ after 40 min of incubation. The reaction mixture contained 0.4 μM GlnJ-UMP, 1.3 μM GlnD, 3 mM MnCl₂, 500 μM α-ketoglutarate, 2 mM ATP, and 0 to 10 mM glutamine. Samples were withdrawn from reaction mixtures after time zero (lanes 1, 3, 5, and 7) or 40 min (lanes 2, 4, 6, and 8). Reactions were stopped by addition of SDS cocktail.

FIG. 9.

Effect of GlnB on deuridylylation. Reaction mixtures contained 1.3 μM GlnD, 3 mM MnCl₂, 500 μM α-ketoglutarate, 2 mM ATP, 10 mM glutamine, and 0.5 mM UTP supplemented with [α-³²P]UTP. Lanes 1 and 2 also contained 0.4 μM GlnB and 0.4 μM His-GlnB-UMP, while only His-GlnB-UMP was added in lanes 3 and 4. Samples were withdrawn from the reaction mixtures after 5 min (lanes 1 and 3) or after 60 min (lanes 2 and 4). Note that both experiments were performed under deuridylylation conditions (1.3 μM GlnD and 10 mM glutamine) but with 0.5 mM UTP supplemented with [α-³²P]UTP also included in the assay. Samples were subjected to SDS-PAGE and visualized by autoradiography.