Characterisation of a novel family of eukaryotic ammonium transport proteins.

Abstract

Species from the family Leguminosae are able to survive in nitrogen (N) limiting conditions via a symbiotic relationship with soil-borne N₂-fixing bacteria collectively known as Rhizobium. The symbiosis results in the development of the root nodule where invaded bacteria (bacteroids) reside within a plant derived membrane vesicle(symbiosome) located within the cytoplasm of infected nodule cortical cells. Bacterial nitrogenase activity converts atmospheric N₂ to ammonium (NH₄⁺), which is delivered to the plant in exchange for photosynthetically derived carbon for bacterial consumption. The mechanism regulating the transfer of NH₄⁺ to the plant across the symbiosome membrane is currently unknown. GmSAT1 (Glycine max Symbiotic Ammonium Transporter 1), a symbiosome membrane bound basic Helix-Loop-Helix (bHLH) transcription factor has previously been identified in soybean by its ability to enhance NH₄⁺ and MA transport in the NH₄⁺ transport deficient yeast strain 26972c (Kaiser et al., 1998). In this study, we have revisited microarray analysis of 26972c cells expressing GmSAT1 to identify differentially regulated yeast genes with putative roles in NH₄⁺/MA transport. Central to this study is the identification of ScAMF1 (Saccharomyces cerevisiae Ammonium Major Facilitator 1), a previously uncharacterised major facilitator transport protein, which was upregulated 56.5-fold in response to GmSAT1 activity. ScAMF1 and GmAMF1;3, a representative AMF1 from soybean, were functionally analysed with respect to putative NH₄⁺ transport using a combination of yeast and Xenopus laevis oocyte expression systems. Both AMF1 proteins enhanced ¹⁴C-MA uptake and established a related sensitivity phenotype in 26972c and 31019b, an alternative NH₄⁺ transport mutant strain. In the presence of low (1 mM) NH₄⁺, ScAMF1 overexpression partially rescued growth of 26972c but was unable to establish a similar phenotype in 31019b. The role of ScAMF1 in NH₄⁺ transport was less clear. However, this study reaffirmed endogenous high-affinity NH₄⁺ transporters called MEPs (Methylammonium Permeases) play an important role in GmSAT1-mediated NH₄⁺ complementation. Heterologous expression in X. laevis oocytes suggest that ScAMF1 and GmAMF1;3 behave as non-selective cation channels capable of low-affinity NH₄⁺ transport, revealing NH₄⁺ current activation by Pi or a product of P-metabolism and potential Ca²⁺-gating. This study also provided a preliminary electrophysiological profile of the Arabidopsis AMF1 homologs with respect to NH₄⁺ transport for future studies to explore in detail.