In vitro interaction between the ammonium transport protein AmtB and partially uridylylated forms of the PII protein GlnZ

Abstract: The ammonium transport family Amt/Rh comprises ubiquitous integral membrane proteins that facilitate ammonium movement across biological membranes. Besides their role in transport, Amt proteins also play a role in sensing the levels of ammonium in the environment, a process that depends on complex formation with cytosolic proteins of the PII family. Trimeric PII proteins from a variety of organisms undergo a cycle of reversible posttranslational modification according to the prevailing nitrogen supply. In proteobacteria, PII proteins are subjected to reversible uridylylation of each monomer. In this study we used the purified proteins from Azospirillum brasilense to analyze the effect of PII uridylylation on the protein''s ability to engage complex formation with AmtB in vitro. Our results show that partially uridylylated PII trimers can interact with AmtB in vitro, the implication of this finding in the regulation of nitrogen metabolism is discussed. We also report an improved expression and purification protocol for the A. brasilense AmtB protein that might be applicable to AmtB proteins from other organisms. [Copyright &y& Elsevier]