Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis

Abstract

PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.

FIG. 1.

(A) Levels of expression of GAL1:HIS3, GAL2:ADE2, and GAL1:lacZ in Y187/PJ696 diploids conferred by different fusion proteins. The relevant medium composition is indicated at the bottom. The photographs were taken 4 days after replica plating (−His and −Ade) or 4.5 h after addition of X-Gal (X-Gal). The vertical arrow indicates decreasing strength of the signals from interacting pairs. The plus and minus signs on the right indicate the relative strengths of the signals based on visual analysis. (B) Interactions involving PII proteins and receptors. (C) Effect of T-loop point mutations in Ser49 on interactions with NAGK. (D) Interactions among PII and/or NAGK fusion proteins. The arrows indicate the orientation of each Y2H interaction detected and point from the GAL4BD construct to the GAL4AD construct. The number of arrowheads indicates the relative strength of the interaction, as shown in panel A. The following color code is used: orange, P. purpurea; green, A. thaliana; white, Synechococcus sp. strain PCC 7942; and grey, E. coli.

FIG. 2.

Comparison of the deduced amino acid sequences of Synechococcus sp. strain PCC 7942 NAGK and representative bacterial and plant proteins. Regions conserved in all sequences are shaded. The asterisks indicate conservation in cyanobacterial and eukaryotic proteins. P. marinus, Prochlorococcus marinus; O. sativa, Oryza sativa; C. tepidum, Chlorobium tepidum; P. aeruginosa, Pseudomonas aeruginosa.

FIG. 3.

Comparison of the deduced amino acid sequences of T loops from PII proteins of different organisms. The positions discussed in the text are shaded. The numbering is the numbering for Synechococcus sp. strain PCC 7942 PII. T. elongatus, Trichodesmium elongatus; T. erythraeum, Trichodesmium erythraeum; P. marinus, Prochlorococcus marinus.

FIG. 4.

Effect of PII on NAGK activity: NAGK assays with permeabilized cells of Synechococcus sp. strain PCC 7942 (WT), a PII knockout mutant (MP2) (glnB⁻), and a derivative strain expressing the PII^S49A protein (MP2-A) (glnB^S49A). Cells were grown in ammonium-containing medium. The average values from three independent experiments are expressed in enzymatic units per milligram of chlorophyll (Chla).