Haloferax mediterraneiGlnK proteins are post‐translationally modified by uridylylation

Abstract

In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2 ), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family.

Effects of T‐loop modification on the PII‐signalling protein: structure of uridylylated Escherichia coli GlnB bound to ATP

“…The uridylylated form of EcGlnB activates the deadenylylating activity of another bifunctional enzyme, glutamine synthetase adenylyl transferase (ATase) (Jaggi et al, 1997;Jiang et al, 2007), which removes the covalently bound AMP from GS, rendering this last enzyme less susceptible to feedback inhibition and therefore more active (Stadtman, 2001). Uridylylation (or adenylylation) of the PII T-loop, originally reported in enterobacteria, occurs in proteobacteria and actinobacteria, and, sporadically, in a few other diverse genera, judged from the conservation of Tyr51 among PII proteins and the occurrence of EcGlnD orthologues (Merrick, 2015), although it might be even more widespread, since it was found in an archaea where a glnD orthologue was not identified (Pedro-Roig et al, 2013). In some unicellular cyanobacteria phosphorylation of a nearby T-loop serine (Ser49) occurs under conditions of nitrogen starvation and may be important for nitrogen regulation in these organisms (Forchhammer and Tandeau de Marsac, 1995).…”

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

“…Recent studies have determined that the PII homologues in the halophilic archaeon Haloferax mediterranei are also subject to this kind of modification (Pedro‐Roig et al. ).…”

“…In proteobacteria, PII proteins are also regulated by UMP modification (uridylylation) as catalyzed by the bifunctional GlnD (uridylyltransferase/uridylyl-removing) enzyme in response to nitrogen availability (Mangum et al 1973;Adler et al 1975;Jiang et al 1998;Zhang et al 2010). Recent studies have determined that the PII homologues in the halophilic archaeon Haloferax mediterranei are also subject to this kind of modification (Pedro-Roig et al 2013a).…”

The structure of a PII signaling protein from a halophilic archaeon reveals novel traits and high‐salt adaptations

“…), Tyr62 has its phenolic group buried, clamped between two residues at the beginning of the loop (Arg47 and Gln50) and forming a hydrogen bond via its phenolic OH with the main chain O atom of Gly48. If this tyrosine is uridylylated in H. mediterranei PII, as concluded from proteomics results , the position of the helical T‐loop observed in our structure, and even the helical folding, might not be possible for the covalently modified T‐loop.…”

“…The observation of clear‐cut putative Shine–Dalgarno sequences preceding the initial ATG codons of the hmglnK1 and hmglnK2 genes (respectively, GGAGG at bases −10 to −14 and AGGAGG at bases −12 to −17 ), and the results of fingerprinting proteomics studies of hmGlnK1 and hmGlnK2 isolated from H. mediterranei , indicate that the N‐terminal extension is indeed present in the naturally produced hmGlnKs. In fact, we found more PII proteins with putative N‐terminal extensions in sequence databases (Table S1), including PII proteins from halophilic organisms.…”