Small protein mediates inhibition of ammonium transport in Methanosarcina mazei – an ancient mechanism?

Small ORF encoded proteins, with a length less than 70 amino acids have been overlooked for long in classical bioinformatics and biochemical approaches. In the past decade however, modern genomics and transcriptomic technologies have discovered small genes containing short open reading frames in many prokaryotic genomes. By differential RNA sequencing, 1340 small open reading frames have been identified in the model organism Methanosarcina mazei, of which 72 have been verified by Mass Spectrometry.

While several bacterial small proteins have already been described, the number of identified and especially functionally characterized small proteins in archaea is still limited. We have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating the nitrogen metabolism in M. mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency. Through in vivo experiments, we have observed that during nitrogen limitation, sP36 is dispersed throughout the cytoplasm; however, upon shifting the cells to nitrogen sufficiency, it relocates to the cytoplasmic membrane. Moreover, in vitro biochemical analysis clearly showed that sP36 interacts with high-affinity with the ammonium transporter AmtB1 (KD=0.26 µM) present in the cytoplasmic membrane during nitrogen limitation, as well as with the PII-like protein GlnK1 (KD=1.8 µM). We propose that in response to an ammonium up-shift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1. This mechanism represents a fast and reversible archaeal way of AmtB regulation, in contrast to the well-studied regulation in bacteria, which depends on covalent modification of GlnK. We will in addition elaborate on the molecular sensing mechanism by sP36 and the observed correlation of sP36 presence and GlnD absence in archaea.