Effects of Sodium and Amino Acid Substrate Availability upon the Expression and Stability of the SNAT2 (SLC38A2) Amino Acid Transporter

Abstract

The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AAs) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis, and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that while AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (N-methylaminoisobutyric acid and glutamine), but not non-substrates. This stabilization and substrate-induced repression were critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteasome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.

TFEB controls retromer expression in response to nutrient availability

“…Retromer-mediated sorting is required as part of an adaptive response to glutamine deprivation To date, mammalian studies of the role of retromer in the sorting of nutrient transporters have primarily focused on cells grown in nutrient-rich conditions (Steinberg et al, 2013;Kvainickas et al, 2017;Yang et al, 2018). For glutamine transporters, nutrient withdrawal induces an adaptive response in which transporters reconfigure their cell surface expression to scavenge extracellular amino acids in an attempt to rebalance nutrient supply with cellular demand (Hyde et al, 2001;Nardi et al, 2015;Bröer et al, 2018;Hoffmann et al, 2018). We therefore subjected wild type and retromer knockout cells to either complete amino acid removal (Earle's balanced salt solution [EBSS]) or the selective removal of glutamine and examined the resulting effects on glutamine transporter trafficking.…”

Molecular physiology of chemical defenses in a poison frog

“…In the liver, the other small molecule transport protein differentially expressed between groups was slc38a2 (also called SNAT2), which had increased transcript abundance in wild frogs compared with laboratory frogs. This protein functions as a sodiumdependent amino acid transporter and its expression is heavily regulated by nutritional factors, where amino acid withdrawal induces slc38a2 expression in vitro (Hoffmann et al, 2018). As ants have lower amino acid content within a hard exoskeleton compared with softer fruit flies and crickets (McCusker et al, 2014), slc38a2 expression in these ant-specialist frogs is likely correlated with diet variation between frog groups rather than the presence of alkaloids.…”

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells

“…Moreover, regulating the cell content of amino acids, SNAT transporters account for the ability of several mesenchymal cell types to recover cell volume under hypertonic conditions [25][26][27][28]33]. Upon amino acid starvation, SNAT2 [23,34] undergoes a marked up-regulation, which is an integral part of the cell response to nutritional stress [35]. As a consequence of the low expression of the SNAT transporters, these characteristic operational features of System A are hindered or even suppressed in MSC, with a blunted increase in substrate uptake upon amino acid starvation (Figure 4), a reduced ability to counteract cell shrinkage upon hypertonic stress ( Figure 6) and lowered intracellular levels of glutamine at the steady state (Figure 1b).…”