RESUMO
Although PII signal transduction proteins have been described in bacteria, archaea and higher plants, no PII homolog has so far been characterized in green algae. In the unicellular green alga Chlamydomonas reinhardtii, the PII protein is encoded by a single nuclear gene CrGLB1. The C. reinhardtii PII (CrPII) was cloned and overexpressed with a C-terminal-fused Strep-tag II peptide. Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of C. reinhardtii to be a homotrimer. Under the studied culture conditions, CrPII appears not to be modified by phosphorylation. Here we show that like its plant PII homologs, the CrPII protein is localized in the chloroplast. Although the CrGLB1 transcript level increased in response to dark-light shift and nitrogen depletion, the level of mature CrPII protein did not change accordingly. Changes in the level of CrGLB1 mRNA were independent of gametogenesis. Characterization of PII in the green alga C. reinhardtii provides a framework for a more complete understanding of the function of this highly conserved signaling protein.
Assuntos
Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Perfilação da Expressão Gênica , Proteínas PII Reguladoras de Nitrogênio/análise , Proteínas PII Reguladoras de Nitrogênio/genética , Sequência de Aminoácidos , Chlamydomonas reinhardtii/fisiologia , Cloroplastos/química , Clonagem Molecular , Escuridão , Expressão Gênica , Luz , Dados de Sequência Molecular , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/química , Multimerização Proteica , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Transdução de SinaisRESUMO
A straightforward protocol for the site-specific incorporation of a 19F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) that incorporates L-4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.
Assuntos
Flúor/análise , Halogenação , Ressonância Magnética Nuclear Biomolecular/métodos , Engenharia de Proteínas/métodos , Oxirredutases do Álcool/análise , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Isótopos , Nitrorredutases/análise , Nitrorredutases/química , Nitrorredutases/genética , Proteínas PII Reguladoras de Nitrogênio/análise , Proteínas PII Reguladoras de Nitrogênio/química , Proteínas PII Reguladoras de Nitrogênio/genética , Plasmídeos/química , Plasmídeos/genética , Salmonella typhimurium/genéticaRESUMO
The GlnK and GlnB proteins are members of the pII signal transduction protein family, which is essential in nitrogen regulation due to this protein family's ability to sense internal cellular ammonium levels and control cellular response. The role of GlnK in nitrogen regulation has been studied in a variety of bacteria but previously has been uncharacterized in the purple nonsulfur anoxygenic phototropic bacterium Rhodopseudomonas palustris. R. palustris has tremendous metabolic versatility in its modes of energy generation and carbon metabolism, and it employs a sensitive nitrogen-ammonium regulation system that may vary from that of other commonly studied bacteria. In R. palustris, there are three annotated forms of pII proteins: GlnK1, GlnK2, and GlnB. Here we describe, for the first time, the characterization of GlnK1, GlnK2, and GlnB modifications as a response to nitrogen availability, thereby providing information about how this bacterium regulates the AmtB ammonium transporter and glutamine synthetase, which controls the rate of glutamate to glutamine conversion. Using a strategy of creating C-terminally tagged GlnK and GlnB proteins followed by tandem affinity purification in combination with top-down mass spectrometry, four isoforms of the GlnK2 and GlnB proteins and two isoforms of the GlnK1 protein were characterized at high resolution and mass accuracy. Wild-type or endogenous expression of all three proteins was also examined under normal ammonium conditions and ammonium starvation to ensure that the tagging and affinity purification methods employed did not alter the natural state of the proteins. All three proteins were found to undergo uridylylation under ammonium starvation conditions, presumably to regulate the AmtB ammonium transporter and glutamine synthetase. Under high-ammonium conditions, the GlnK1, GlnK2, and GlnB proteins are unmodified. This experimental protocol involving high-resolution mass spectrometry measurements of intact proteins provides a powerful method of examining the posttranslational modifications that play a crucial role in both the regulation of the AmtB ammonium transporter and glutamine synthetase within R. palustris.
