Spatial regulation of the mTORC1 system in amino acids sensing pathway

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine protein kinase that regulates numerous cellular processes including cell growth, proliferation, cell cycle, and autophagy. mTOR forms two different multi-protein complexes referred to as mTOR complex 1 (mTORC1) and mTORC2, and each complex exerts distinct functions exclusively. mTORC1 activity is sensitive to the selective inhibitor rapamycin, whereas mTORC2 is resistant. mTORC1 is regulated by many intra- and extra-cellular cues such as growth factors, nutrients, and energy-sensing signals, while mTORC2 senses ribosome maturation and growth factor signaling. This review focuses on current understandings by which mTORC1 pathway senses cellular nutrient availability for its activation.     

Differential regulation of amino acid transporter SNAT3 by insulin in hepatocytes

The liver is a metabolism and transfer center of amino acids as well as the prime target organ of insulin. In this report, we characterized the regulation of system N/A transporter 3 (SNAT3) in the liver of dietary-restricted mice and in hepatocytes treated with serum starvation and insulin. The expression of SNAT3 was up-regulated in dietary-restricted mice. The expression of SNAT3 protein was detected on the plasma membrane of hepatocyte-like H2.35 cells with a half-life of 6-8 h. When H2.35 cells were depleted of serum, the expression of SNAT3 was increased. An increased concentration of insulin, however, suppressed SNAT3 expression. Interestingly, the down-regulation of SNAT3 expression by insulin was blocked by the specific phosphoinositide 3-kinase inhibitor LY294002 and mammalian target of rapamycin inhibitor, but not by MAPK inhibitor PD98059, suggesting that insulin exerts its effect on SNAT3 through phosphoinositide 3-kinase-mammalian target of rapamycin signaling. Surface biotinylation assay showed an increased level of SNAT3 on the cell surface after 0.5 h of insulin treatment, although no effect was observed after 24 h of treatment. Consistently, the transport of the substrate l-histidine was increased with short, but not long, treatment by insulin in both H2.35- and SNAT3-transfected COS-7 cells. The L-histidine uptake was inhibited significantly by L-histidine followed by 2-endoamino-bicycloheptane-2-carboxylic acid and L-cysteine and to a lesser extent by L-alanine and aminoisobutyric acid, but was not inhibited by alpha-(methylamino)isobutyric acid, implying that uptake of L-histidine in H2.35 cells is primarily mediated by system N transporters. In conclusion, differential regulation of SNAT3 by insulin and serum starvation reinforces the functional significance of this transporter in liver physiology.     

Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids

Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.