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.
Highlights
The System A amino acid (AA) transporter family is comprised of three isoforms, SNAT1, 2, and 4, of which SNAT2 (SLC38A2) is the most widely expressed and most extensively regulated (Mackenzie and Erickson, 2004)
Since expression of SNAT2V5 is driven by an AA insensitive CMV promoter and transfection efficiency between cells incubated in buffer containing or lacking AAs was similar within each experiment, the elevated SNAT2V5 abundance induced upon AA deprivation is likely to reflect increased stabilization of the carrier. This latter finding is consistent with previous work from our lab (Hyde et al, 2007, 3143 /id), but we show here that the increase in SNAT2V5 is associated with a modest (∼30%) increase in methylaminoisobutyric acid (Me-AIB) uptake over and above that recorded in control cells transfected with the empty vector, in which, the elevated Me-AIB uptake elicited by AA deprivation is due to the adaptive upregulation of the endogenous SNAT2 transporter (Figure 2C)
Since the 7A-SNAT2V5 protein is stable under the different conditions tested, we argue that binding of AA substrates to the native transporter may induce a conformational change in the protein that make one or more of the target lysyl residues in the N-terminal SNAT2 domain accessible for ubiquitination by its E3-ligase that promotes ubiquitinated SNAT2 internalization/degradation
Summary
The System A amino acid (AA) transporter family is comprised of three isoforms, SNAT1, 2, and 4, of which SNAT2 (SLC38A2) is the most widely expressed and most extensively regulated (Mackenzie and Erickson, 2004). The secondary active transport of extracellular AAs results in an outwardly directed concentration gradient of SNAT substrates that we have shown can subsequently exit the cell via tertiary exchange transporters, such as System L, which operate alongside SNAT carriers in the plasma membrane (Baird et al, 2009; Hundal and Taylor, 2009) This tertiary exchange coupling is of particular significance as it influences the intracellular delivery of essential AAs (e.g., leucine) that have a potent stimulatory effect on mTORC1, a signaling complex regulating key cellular processes such as mRNA translation, cell growth/metabolism, and autophagy (Saxton and Sabatini, 2017). It follows that factors affecting expression and activity of SNAT2 may impact on mTORC1 activation and regulation of these latter processes (Pinilla et al, 2011; Uno et al, 2015)
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