N-Acetyl-L-glutamate Kinase of Chlamydomonas reinhardtii: In Vivo Regulation by PII Protein and Beyond

Abstract

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

Keywords: N-Acetyl-L-glutamate kinase; PII- signal transduction protein; arginine biosynthesis; green algae.

Figure 1

Effects of ammonium and nitrate on cell growth and relative CrNAGK1 gene expression. (a) The growth curves were analyzed in the presence of 7.5 mM NH₄Cl or 4 mM KNO₃. Values are means ± SE of three independent experiments; (b) Time course of the CrNAGK1 transcripts accumulation during growth of cells in ammonium- or nitrate-containing medium. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a housekeeping gene RACK1.

Figure 2

Effects of ammonium and nitrate on the specific activity of CrNAGK and the total free content of Arg and Gln. (a) Time course of the CrNAGK activity during growth of cells in ammonium- or nitrate-containing medium. Cells were grown as described in Figure 1a; (b) Relationship between nitrogen source and intracellular Arg content; (c) Relationship between nitrogen source and intracellular Gln content. In (b,c), the content of amino acids is expressed in µg per10⁶ cells. Values are means ± SE of three biological replicates.

Figure 3

Effects of nitrite on cell growth, CrNAGK expression and activity, and the total free content of Arg and Gln. (a) The growth curve was analyzed in the presence of 10 mM KNO₂. Values are means ± SE of three independent experiments; (b) Time course of the CrNAGK1 transcripts accumulation during growth of cells in nitrite-containing medium. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a housekeeping gene RACK1; (c) Time course of the CrNAGK activity during growth of cells in nitrite-containing medium; (d) Relationship between nitrogen source and intracellular Arg and Gln content. The concentration of amino acids is expressed in µg per10⁶ cells. Values are means ± SE of three biological replicates.

Figure 4

Characterization of amiRNA-GLB1 strains. (a) CrPII abundance in wild-type strains (CC4533 and CC3491), amiRNAGLB1-65, and amiRNAGLB1-88. Protein levels were analyzed by Western blotting. Each line corresponds to 40 μg of soluble proteins extracted from samples taken from cultures incubated in nitrite-containing medium for 24 h. GLB1-66 and GLB1-88 indicate amiRNAGLB1-65 and amiRNAGLB1-88, respectively; (b) RT-qPCR analysis of CrGLB1 transcript levels. Relative expression levels were normalized with the gene expression of RACK1 and calculated using ΔCT. Samples were analyzed from cultures incubated in ammonium- or nitrite-containing medium for 24 h. Values are means ± SE of three biological replicates and three technical replicates.

Figure 5

Effects of ammonium and nitrite on CrNAGK expression and activity, and the total free content of Arg in CC3491 and GLB1-knockdown strains. (a,b) Time course of the CrNAGK activity during growth of cells in ammonium- or nitrite-containing medium; (c,d) Time course of the CrNAGK1 transcripts accumulation during growth of cells in ammonium- or nitrite-containing medium. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a housekeeping gene RACK1; (e,f) Relationship between nitrogen source and intracellular Arg content. Intracellular Arg concentration at 0 h in each strain is considered as control (set to 100%). Values are means ± SE of three biological replicates. * and ** denote significant differences between parental strain and CrGLB1-underexpressing transformants according to the Student t-test (p-value < 0.01 or <0.05, respectively).

Figure 6

Effects of N deprivation on CrNAGK expression and activity, and the total free content of Arg in wild types and GLB1-knockdown strains. (a) CrNAGK activity and intracellular Arg content in 6145c strain during incubation in N-free medium; (b) Time course of the CrNAGK activity in parental strain and amiRNAGLB1 strains during incubation in N-free medium. * denotes significant differences between parental strain and CrGLB1-underexpressing transformants according to the Student t-test (p-value < 0.01 or <0.05, respectively, (c) Intracellular Arg content in parental strain and amiRNAGLB1 strains during incubation in N-free medium. Arg concentration in each strain in N-replete medium is considered as control (set to 100%). Values are means ± SE of three biological replicates; (d) Time course of the CrNAGK1 transcripts accumulation in parental strain and amiRNAGLB1 strains during incubation in N-free medium. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a housekeeping gene RACK1.

Figure 7

Anticipated model of CrNAGK regulation under N-sufficiency and N-limitation. When N is available, the L-glutamine concentration increases, resulting in PII-NAGK complex formation. Under these conditions, PII alleviates NAGK from Arg feedback inhibition and thereby enhances NAGK activity and Arg production. In addition, nitrate and nitrite upregulate NAGK1 and GLB1 gene expression. When cells became N deficient for a short period of time, an increase in NAGK and PII levels appears to be enough to contribute to NAGK-PII complex formation and keep the enzyme active, which in turn results in elevated Arg synthesis. Under N-limitation for a long period of time, NAGK activity decreases and becomes the lowest. This could be achieved by releasing signal protein from the complex with NAGK through a reduction in Gln content and/or PII sequestration by an additional target. The release of PII results in stronger arginine feedback inhibition of NAGK, diminishing energy consumption and flux into arginine. An additional mechanism responsible for negative control of NAGK at the post-translational level is proposed. The positive transcriptional regulation is indicated by (+). The width of the green arrows is indicative of the levels of Arg biosynthesis. Blunted lines denote the negative regulation at the post-translational level.