Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Abstract

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

Sound of silence: the beauvericin cluster in Fusarium fujikuroi is controlled by cluster‐specific and global regulators mediated by H3K27 modification

“…Several cross‐linking technologies have been developed to demonstrate the physical interaction between transporters and the respective kinases (Stolarczyk et al ., ). For the general amino acid (Gap1) and ammonium (Mep2) permeases in yeast it has been shown that they are involved in substrate sensing and regulating cellular processes by their interaction with protein kinases and the respective signaling cascades (van den Berg et al ., ). However, a similar function as sensor and regulator has never been demonstrated for an ABC transporter.…”

Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor

“…Fungal Mep proteins of the Mep-Amt-Rh family possess 11 TM domains with a periplasmic N-terminal domain and a cytoplasmic CTD ( Fig 1A) [15,19]. The transport activity of the The ammonium transport protein Mep2 is regulated by its C-terminal extremity and the Npr1 kinase.…”

“…Structural data support a modified Transport activity of Mep2 CTD variants correlates with pseudohyphal growth efficiency. Homozygous diploid triple-mepΔ (ZAM38) and triple-mepΔ npr1-1 (ZMB058) cells were transformed with the pFL38 empty plasmid (-) or with YCpMep2, YCpMep2 S426stop , YCpMep2 CΔ428-431 , YCpMep2 CΔ428-449 , YCpMep2 CΔ434-449 , YCpMep2 CΔ434-485 , YCpMep2 CΔ442-449 , YCpMep2 CΔ442-485 , YCpMep2 or YCpMep2 conformation of the CTD of a Candida albicans Mep2 variant with analogous phosphomimetic environment [15]. We took advantage of the S457D mutation to test whether a Mep2 protein locked in a putative active fold allows filamentation even in the absence of ammonium limitation.…”

“…S. cerevisiae possesses three members of the conserved Mep-Amt-Rh protein family mediating the transmembrane transport of ammonium, hereafter referring to the sum of NH 4 + and NH 3 molecular entities [11][12][13][14]. The proteins of this family, comprising the human Rhesus factors, display 11 or 12 transmembrane (TM) spans and a cytosolic C-terminal domain (CTD), and adopt a trimeric fold [15][16][17][18][19]. Of the three yeast MEP genes, only the specific deletion of MEP2 impairs filamentation, growth being ensured by the ammonium transport activity of the remaining Mep1 and Mep3 paralogues [20].…”

A conformational change in the N terminus of SLC38A9 signals mTORC1 activation

“…The N-terminus of SLC38A9, om the other hand, was previously shown to interact directly with the Rag-Regulator complex to activate mTORCl (16) . Collectively, these results suggest that SLC38A9 is a “transceptor”, which is membrane protein that embodies the functions of both a transporter and a receptor (23–27) .…”