Amino Acid Starvation Induces the SNAT2 Neutral Amino Acid Transporter by a Mechanism That Involves Eukaryotic Initiation Factor 2α Phosphorylation and cap-independent Translation

Abstract

Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2␣ that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2␣ phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2␣ phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2␣ phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.The severe nutritional stress caused by amino acid starvation triggers several adaptive changes. One of these is the stimulation of transport system A for neutral amino acids. Adaptive regulation of system A has been described in most amino acid-starved animal cells (1-3). Work from several laboratories (4 -6) has demonstrated that this regulatory mechanism is associated with an increase in the mRNA for SNAT2, the ubiquitously expressed member of the sodium-coupled neutral amino acid transporter family (7). The induction of SNAT2 transcription leads to the increased synthesis of SNAT2 carriers, which are found in greater amounts on the membranes of starved cells (5,8). Recently, an amino acid-responsive element has been identified in the first intron of the mouse and human SNAT2 genes (9), supporting the hypothesis that transcriptional activation contributes to the adaptive regulation of system A.Unlike transcriptional activation, the translation of SNAT2 during amino acid starvation has received little attention ...

Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling

“…Activation of this pathway appears to depend on the overall level of cellular stress imposed and, perhaps surprisingly, is usually more pronounced when a single amino acid is withdrawn than when total amino acid starvation is imposed. Moreover, the finding that the 5Ј-UTR of the SNAT2 mRNA contains an internal ribosome entry sequence (IRES) suggests that SNAT2 mRNA will be efficiently translated via a cap-independent mechanism despite any global reduction in protein synthesis that would be associated with amino acid starvation (37). Notwithstanding the important contribution that these latter processes will make to an increase in System A expression/activity, there is also mounting evidence that interactions between SNAT2 and its substrates may play an important role in the adaptation response.…”

Reversible induction of translational isoforms of p53 in glucose deprivation

“…21,22 Amino acid depletion induces GCN2 kinase-mediated phosphorylation of eIF2α, leading to a global decrease in protein synthesis and induction of an adaptive survival program. 21,23 Under this condition, IRES-mediated translation of the Cat-1 and SNAT2 mRNAs occur, 24,25 thus preparing cells to transport amino acids once they become available. Methionine synthase, a key enzyme that clears intracellular homocysteine, is induced by its cofactor, vitamin B12, at a translational level through an IRES in the 5'UTR of the mRNA.…”

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

“…These data indicate that the yrpS5 substitution did not specifically affect URE2 IRES-mediated expression. To investigate the activities of two mammalian IRESs in yeast, the SNAT-2 IRES (Gaccioli et al 2006) and the CAT-1 IRES (Fernandez et al 2001;Yaman et al 2003), these IRESs were cloned into the p281-4 vector behind the stable hairpin structure described above and fused to the lacZ reporter gene. The SNAT-2 and the CAT-1(-192) IRESs (Yaman et al 2003) were active in the WT yeast strain (Fig.…”

“…The CAT-1(-192) IRES (Yaman et al 2003) was amplified by PCR using 59-AAAAACTCGAGCCTTGCAGGGGCGTGA AGCTACT-39 and 59-AAAAAGAATTCTCATCGCGCTGAGCAA ATCTGTCTG-39 primers. The SNAT-2 IRES (Gaccioli et al 2006) was amplified using 59-AAAAACTCGAGCGACGCCGCCGCCT TAGAAC-39 and 59-AAAAAGAATTCTCATGCTAAGCACTGGG AGGAATCGG-39 primers. PCR fragments were digested with XhoI and EcoRI and were cloned into the p281-4 vector.…”