Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Abstract

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.

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

“…As for several Mep2 variants, we observed an increased filamentation efficiency in cells lacking Npr1, we evaluated whether the kinase absence could per se enhance filamentation, independently of Mep2 in triple-mepΔ npr1-1 cells. We previously showed that overexpression of Mep1 in Npr1-lacking cells is sufficient to bypass the kinase requirement and allow Mep1 activity [40]. Consistently, Mep1 overexpression enabled growth of triple-mepΔ npr1-1 cells on SLAD and SHAD ( Fig 4B).…”

“…Available experimental data suggest that, following initial recognition, NH 4 + undergoes deprotonation and NH 3 then permeates through the hydrophobic pore while the fate of the released proton remains unclear [18,[32][33][34][35][36][37][38]. A difference between Mep1-and Mep2-type proteins has already been depicted and notably concerns the molecular mechanisms enabling the activity tuning by the TORC1 effector kinase Npr1 [39][40][41]. The kinase mediates the S457-phosphorylation and thereby the silencing of an autoinhibitory CTD in the case of Mep2 and controls the phosphorylation of an intermediate inhibitory partner, Amu1/Par32, in the case of Mep1 as well as its close paralogue Mep3 [39][40][41].…”

“…A difference between Mep1-and Mep2-type proteins has already been depicted and notably concerns the molecular mechanisms enabling the activity tuning by the TORC1 effector kinase Npr1 [39][40][41]. The kinase mediates the S457-phosphorylation and thereby the silencing of an autoinhibitory CTD in the case of Mep2 and controls the phosphorylation of an intermediate inhibitory partner, Amu1/Par32, in the case of Mep1 as well as its close paralogue Mep3 [39][40][41]. We show here that filamentation is intimately related to Mep2 transport activity.…”

Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation

“…Even though the connection between TOR activity and nitrogen uptake and assimilation seems to be similar between distantly related model species, the mechanistic implementation appears quite different. While yeast directly regulates the ammonium transporter in two Npr1-dependent pathways (Boeckstaens et al, 2014(Boeckstaens et al, , 2015, mammalian cell cultures have a more indirect Npr1-dependent nitrogen-recycling mechanism, which relies on endocytosis and ubiquitin-based protein degradation (MacGurn et al, 2011). The direct mechanistic elucidation of this signaling cascade starting from TOR inhibition to ammonium uptake and assimilation followed by amino acid sensing in a tractable green model species like Chlamydomonas will therefore allow us to close the gap between yeast, mammals, and photosynthetic organisms, providing further dimensions to the field of growth signaling in various eukaryotes.…”

Pib2 and the EGO complex are both required for activation of TORC1

“…One additional target of active Npr1 is the poorly characterized protein Par32. Active Npr1 results in extensive phosphorylation of Par32 at multiple sites, which leads to a significant change in migration rate (Boeckstaens et al, 2015). Therefore, the migration rate of Par32 can be used as a readout to evaluate the activity of Npr1.…”