In this study we aimed to systematically screen the yeast genome for genes necessary in sensing amino acid starvation. To achieve this goal, we first established a robust flow cytometry-based assay to monitor the expression of a TOR-sensitive transcriptional reporter. We believe a GFP-based readout has three advantages over a luciferase-based readout: (1) by employing GFP, there is no need to lyse cells; (2) a fluorescence reading can be obtained for each individual cell, thereby abolishing the need to normalize the signal for the number of cells; and (3) this assay offers a possibility of more complicated analyses with sub-populations of cells, such as by co-induction of two different fluorescent proteins. Compared to assays where the plate could be scanned, however, performing flow cytometry in a high throughput manner is still time-consuming. It takes about 45 minutes to process each 96 well plate, with the majority of time spent flushing the fluidics system between samples.
Using this assay, we found an evolutionarily conserved Npr2/3 complex that is responsible for the inactivation of TORC1 in response to amino acid limitation. In response to amino acid withdrawal, cells missing NPR2 and NPR3 fail to completely inactivate such TORC1 readouts as Npr1 phosphorylation, Gat1 inhibition and ribosome biosynthesis. Correspondingly, these cells are unable to adjust to a growth environment where the nitrogen source is limiting or of poor quality. Our data appear to indicate that the Npr2/3 complex specifically mediates a signal about the low quality/quantity of amino acids since phenotypes associated with NPR2/3 deletion are evident only when cells are grown on nitrogen sources that are sub-optimal for TORC1 activity. For instance, when cells are grown with peptone or glutamine as nitrogen sources, TORC1 is fully active and under those conditions npr2Δ and npr3Δ cells do not exhibit proliferation defects. Under conditions where a lower activity of TORC1 is required, such as in the presence of proline or ammonia, Npr2/3 are responsible for lowering this activity. In the simplest model, the Npr2/3 complex senses the levels of glutamine and inactivates TORC1 when those levels are low. Further studies must determine whether the Npr2/3 complex is itself responsible for sensing intracellular amino acid pools or whether this complex simply mediates the signal. Interestingly, our results indicate that neither protein plays a role in carbon starvation, since in both mutants TORC1 is inhibited when glucose is removed from the media and both mutants proliferate normally under glucose-limiting conditions ().
Despite the high degree of conservation, our best efforts to assign an activity or function to either Npr2 or Npr3 were unsuccessful, as neither protein exhibits characteristic domain structures that could predict their cellular function. Our efforts to co-immunoprecipitate TORC1 and Npr2/3 were inconclusive (data not shown). Also, the Tor1/Kog1 complex was unaltered in npr2Δ and npr3Δ cells (data not shown). Further complicating analysis of both proteins is their relatively low abundance in the cell. In fact, in our hands human NPRL2 is detectable in only a few cell lines (data not shown). This has hindered our attempts to purify additional members of the complex, as well as visualize their sub-cellular location. Clearly, further biochemical and genetic approaches are needed to shed light on the mechanistic aspects of their function.
Recently, two groups characterized a role Rag GTPases play in activating TORC1 by amino acids in human tissue culture cells and D. melanogaster 
. These studies demonstrated that Rag GTPases are part of the extended TORC1 complex and that cells expressing constitutively active RagA/B fail to inactivate TORC1 when amino acids are withdrawn. Interestingly, Rag A and RagB are similar to each other and they appear to be orthologus to yeast Gtr1. If yeast Gtr1 was responsible for activating TORC1, then deletion of GTR1
should lead to lethality or at least mimic a rapamycin-like slow proliferation phenotype. Yet, gtr1
Δ cells appear normal and don't express the TORC1-sensitive Dal80pr-GFP reporter in rich media (data not shown). This discrepancy could be explained by either a substitution of Gtr1 by another GTPase in yeast, or by a divergence of the amino acid signaling pathway to TORC1 between metazoans and yeast. By a similar token, yeast Vps34 did not emerge from our screen as a regulator of TORC1 (see Introduction
). Further studies must examine the role of Rag GTPases in lower eukaryotes and whether the Npr2/3 complex is functionally conserved in higher eukaryotes. Keeping with the conserved nature of TORC1 and its regulation by amino acids, it would be exciting if a unifying mechanism emerges.
Except for mTOR itself, many of the upstream and downstream components of the mTOR pathway are known to be either proto-oncogenes or tumor suppressors 
. Interestingly, early reports have implicated NPRL2
as a tumor suppressor gene. NPRL2
is located on the human chromosome 3p21.3 homozygous deletion region 
, a region that is deleted in various human cancers 
. Further, reintroduction of NPRL2
into these cells inhibits cell proliferation both in cell lines and in human lung cancer mouse models 
. Human NPRL2-null cells and yeast npr2
Δ cells both have been shown to exhibit resistance to cisplatin 
, suggesting functional conservation and raising the possibility that NPRL2 might act as a repressor of mTORC1. It remains to be determined whether a mutation in NPRL2
is truly the driver mutation that gives rise to neoplastic growth via overactive mTORC1, but a high mTORC1 activity has certainly been ascribed to invasive tumors 
. Further, as cancer cells with hyperactive mTORC1, either due to loss of PTEN
, have been shown to be particularly sensitive to rapamycin treatment 
, we speculate that cancer cells with mutated NPRL2
might also be particularly sensitive to rapamycin. Also, since both Npr2 and Npr3 are necessary for inactivation of TORC1 in yeast, it would be interesting to examine whether a deletion of human NPRL3
also correlates with tumorigenesis.
In conclusion, here we report the discovery of a novel, conserved Npr2/3 complex that specifically inactivates TORC1 in response to amino acid limitation in yeast. Inactivation of TORC1 is important for adaptation to an environment with scarce amino acids, since diminished TORC1 activity leads to activation of amino acid permeases and catabolic enzymes, repression of ribosomal protein gene expression, and induction of macroautophagy. Cells lacking NPR2 or NPR3 are unable to inactivate TORC1, and they fail to thrive in nitrogen challenged environment as a result. These results demonstrate a unique way by which the cells respond to nutrient limitation, and this complex provides a fertile ground to study the regulation of this important pathway.