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

In the past decade, small open reading frames (sORFs) coding for proteins less than 70 amino acids (aa) in length have moved into the focus of science. sORFs and the corresponding small proteins have been recently identified in all three domains of life. However, the majority of small proteins remain functionally uncharacterized. While several bacterial small proteins have already been described, the number of identified and functionally characterized small proteins in archaea is still limited. In this study, we have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency, as compared to the wild type. Through our 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. Furthermore, an in vitro biochemical analysis clearly showed that sP36 interacts with high affinity with the ammonium transporter AmtB1 present in the cytoplasmic membrane during nitrogen limitation as well as with the PII-like protein GlnK1. Moreover, the in vivo GlnK1 interaction with AmtB1 due to nitrogen upshifts requires the presence of sP36. Based on our findings, we propose that in response to an ammonium upshift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1.

Automated summary

In the past decade, scientists have focused on small open reading frames (sORFs) that code for proteins less than 70 amino acids in length. These sORFs and corresponding small proteins have been found in all three domains of life, but most of them are still not fully understood. This study reveals the critical role of a small protein called sP36, consisting of only 61 amino acids, in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency. Through in vivo experiments, it is observed that sP36 is dispersed throughout the cytoplasm during nitrogen limitation, but relocates to the cytoplasmic membrane upon shifting the cells to nitrogen sufficiency. In addition, in vitro biochemical analysis shows that sP36 interacts with high affinity with the ammonium transporter AmtB1 and the PII-like protein GlnK1 during nitrogen limitation. Moreover, the interaction between GlnK1 and AmtB1 due to nitrogen upshifts requires the presence of sP36.