Examination of quantitative trait loci that regulate chronological aging and telomere length in the progeny from a cross between the laboratory strain S288c and a vineyard strain, RM11-1a, led to identification of a polymorphism in BUL2 which alters trafficking of amino acid permeases and cellular amino acid import. Loss of Bul2 function, conferred by the laboratory allele of the gene, initiates a cascade of events outlined in that centers on TOR, a nutrient-responsive protein kinase previously implicated in CLS control. Our study defines a novel downstream role for TOR signaling in the regulation DNA replication and telomere maintenance through Gln3-mediated assembly of ribonucleotide reductase during S-phase.
Model for BUL2-mediated alteration of telomere length and chronological lifespan.
Amino acids are powerful activators of TOR signaling not only in yeast, but also in multicellular eukaryotes. For Drosophila melanogaster
larvae, amino acid deprivation inhibits TOR activity and leads to growth inhibition and reduced body size 
. Similarly, Caenorhabditis elegans
lacking the intestinal amino acid transporter pep-2 also have reduced body size 
. Increasing evidence suggests that reduced intake of amino acids, which consequently reduces TOR activity, may be a key component of life-extending dietary interventions. Lifespan extension granted by dietary restriction in D. melanogaster
was abolished by re-addition of amino acids 
. Additionally, dietary reduction of a single essential amino acid, either tryptophan or methionine, was sufficient to confer lifespan extension in both mice and rats 
. While dietary restriction studies in S. cerevisiae
typically involve glucose restriction, our finding that restoration of Bul2 function and resulting reduction of cellular amino acid import extends CLS supports the idea that amino acid-mediated regulation of TOR signaling controls longevity.
While several of the upstream molecular events that control TOR activity, such as growth factors and energy status, are understood in great detail 
, we only have rudimentary knowledge of how cells sense amino acid sufficiency and transmit this signal to TOR. TOR forms two separate complexes: the rapamycin-sensitive TOR complex 1 (TORC1), which regulates growth, ribosome biogenesis, translation and lifespan, and the rapamycin-insensitive TOR complex 2 (TORC2) involved in actin cytoskeleton organization and cell wall integrity 
. Recent studies in mammalian cells have identified several components that are required for TOR activation by amino acids, including Rag GTPase heterodimers involved in the recruitment of TORC1 complex to the lysosomal membrane compartment 
. In addition to their roles as activators of TOR, the S. cerevisiae
Rag GTPase orthologs Gtr1 and Gtr2 
are also implicated in the retrieval of Gap1 and other high affinity amino acid permeases from the vacuolar trafficking pathway 
, thus promoting their localization to the plasma membrane. Because the retrieval of Gap1 from the vacuolar targeting pathway is regulated by amino acid availability (discussed below), these findings raise the possibility that the related amino acid-responsive pathway that controls TOR also controls recycling of high affinity transporters to the cell membrane.
In contrast to the majority of the 23 amino acid permeases in yeast, which are constitutively expressed and import specific amino-acids with low affinity, high affinity permeases such as the general amino acid permease Gap1 and proline permease Put4 are highly expressed only during nitrogen limitation 
. Gap1 and its related class of permeases have a high capacity for amino acid transport and are thought to scavenge amino acids for use as a source of nitrogen. Intracellular sorting is one of the mechanisms by which the quality of available nitrogen controls the presence of high affinity permeases at the cell membrane: during growth with a good nitrogen source such as ammonium, glutamate and glutamine, Gap1 is sorted to the vacuole for degradation 
. When cellular nitrogen and amino acids levels are low, Gap1 is sorted to the plasma membrane. A complex consisting of Rps5, Bul1 and Bul2 ubiquitylates Gap1 and specifies its sorting to the multivesicular endosome. From the endosome, Gap1 can be targeted either to the vacuole or trafficked to the plasma membrane depending of the amino acid availability 
. The amino acid-regulated step in this process appears to be Gap1 retrieval from the endosome rather than Gap1 ubiquitylation. Nevertheless, ubiquitylation is a prerequisite for controlling Gap1 localization because in its absence, Gap1 never reaches the endosome and is constitutively targeted to the plasma membrane. Therefore, loss of Bul2 function, such as in cells with the BY allele of BUL2, results in non-discriminatory import of amino acids and greater intracellular amino acid and nitrogen availability. Our finding that the common laboratory strain S288c carries a loss-of-function mutation in BUL2, subsequently leading to indiscriminant amino acid uptake, is important for future studies that exploit yeast as a model for amino acid sufficiency and TOR signaling. Specifically, such studies should include strains with wild-type BUL2; for example, they could employ the allele substitution strains described here. The mutation in BUL2 adds to the list of genetic alterations in the standard laboratory strain that are not representative of other members of the species such as loss of function changes in AMN1 
and MKT1 
Similar to the control of Gap1, mammalian growth factor receptors are also regulated by ubiquitin-mediated trafficking. While yeast cells detect cellular resources directly through their import via permeases, multicellular organisms rely on growth factors such as IGF-1, which also stimulates TOR activity through Akt-Tsc-Rheb signaling, to coordinate nutrient availability with growth 
. Cell surface localization of the IGF-1 receptor (IGF-1R) has been shown to depend on ubiquitylation by Nedd4, a homolog of the catalytic Rsp5 subunit of the Rsp5/Bul1/Bul2 ubiquitin ligase 
. It is intriguing that Nedd4−/+
mice have reduced IGF-1 receptors on the cell surface and phenotypes consistent with reduced IGF-1 signaling, including decreased body size 
, raising the possibility that they may share increased longevity with other IGF-1-related dwarf mice.
Reduced amino acid import in cells with functional Bul2 inhibits TORC1 activity, consistent with our observation of increased activity of TOR-inhibited transcription factor GLN3 in cells containing the RM BUL2 allele compared with cells which have the BY allele of BUL2. (In favorable nitrogen conditions, high TORC1 activity sequesters Gln3 in the cytoplasm.) Reduced TOR activity has been previously shown to extend both chronological and replicative lifespan in yeast 
. Because reduced TOR activity extends lifespan also in higher eukaryotes 
, there is great interest in understanding the downstream events that mediate this effect.
Several mechanisms by which nutrients and TOR inhibition promotes CLS in yeast have been proposed, including reduced accumulation of acetate and/or acidification of culture media 
, promotion of respiration and autophagy 
, and increased activity of stationary phase and stress-responsive transcription factors 
. CLS experiments are often carried out in synthetic media which is complicated by significant media acidification due to release of organic acids during fermentation (the initial media pH of 4.2 decreases to <3 after cells reach stationary phase). A combination of acidic pH and high concentration of acetate in the media has been linked to reduction of cell viability 
. Because our chronological aging assays are performed in rich media (YPD), which has an initial pH of 6.0 that reduces only to 5.8 after cells reach stationary phase, or buffered synthetic media, acetate toxicity is an unlikely mechanism for CLS modulation in our study.
A study by Bonawitz et al.
linked reduction in TOR activity to increased cellular respiratory capacity 
. While translation is generally inhibited by reduced TOR activity, Bonawitz et al.
found that translation of mitochondrial proteins was enhanced and led to increased respiration during growth in glucose. Respiration becomes increasingly important for maintaining energy supplies and viability as cells transition from fermentative growth to stationary phase. The importance of respiration during the stationary phase transition is supported by the findings of two recent genome-wide studies that identified respiratory deficient mutants among those with the shortest CLS 
. In the same studies, mutants defective in autophagy, another process stimulated by TOR inhibition, were also found to have short CLS. These observations suggest that autophagy and respiration constitute important mediators by which reduced TOR activity promotes CLS.
The inhibition of TOR that occurs in cells during the post-diauxic shift and preparation for stationary phase also elicits specific transcriptional responses that are essential for maintaining viability during quiescence 
. One target of TOR is the Rim15 protein kinase that translates nutrient limitation signals from TOR, as well as Ras/PKA and Sch9, into upregulation of cellular responses necessary for survival in stationary phase 
. Similarly to Gln3, Rim15 is phosphorylated by the nutrient-sensing kinases and retained in the cytoplasm, but upon nutrient deprivation, dephosphorylated Rim15 translocates to the nucleus to activate transcription factors Gis1 and Msn2/4, which upregulate genes necessary for post-diauxic shift 
and stress response respectively 
. Deletion of either RIM15 or its target transcription factors shortens CLS and abolishes benefits conferred by caloric restriction or mutations in Tor/Ras/Sch9 that mimic calorie restriction 
. Since Rim15 and Gln3 are both directly regulated by TOR through cytoplasmic sequestration, we predicted that Gln3, like Rim15, would be essential for proper stationary phase transition and survival. In support of this idea, we have found that deletion of GLN3 in the vineyard strain dramatically shortens CLS (Figure S4
) and that alteration of Bul2 function did not affect CLS in gln3Δ
mutants. However, consistent with previous reports 
, we found that deletion of GLN3 in the laboratory strain increased CLS. The paradoxical increase in CLS in response to GLN3 deletion in the laboratory strain is in opposition to the CLS detriment conferred by the loss of function of other transcription factors such as Msn2/4 or Gis1 which are, similarly to Gln3, upregulated during starvation. Furthermore, the opposing effect of GLN3 deletion in the laboratory and vineyard strains makes it difficult to determine the precise role of GLN3 as a mediator of CLS alterations in the cascade of events initiated by the BUL2 polymorphism.
Serving as a central link between nutrient availability and growth, TORC1 regulates many cellular processes including ribosome biogenesis, protein translation, autophagy and respiration 
. During the examination of how telomere maintenance is affected by amino acid import, we discovered that ribonucleotide reductase (RNR) complex assembly during S-phase is modulated by the TOR-responsive transcription factor Gln3, defining a novel downstream role for TOR in DNA replication. We found that increased Gln3 activity, conferred by the deletion of URE2, upregulates Wtm1, which, in turn, promotes nuclear retention of the small RNR4 subunit in the nucleus. Deletion of WTM1 restores cytoplasmic localization of the small subunits and partially rescues the telomere length defect of ure2Δ
cells. TORC1 inhibition by rapamycin was previously associated with genotoxic stress sensitivity and inability to maintain high Rnr1 and Rnr3 levels in response to DNA damage 
. Using telomere length as a phenotype, we have uncovered a role of TORC1-responsive transcription factor GLN3 in modulation of RNR assembly during S-phase in response to cellular amino acid availability. TOR-mediated control of DNA replication adds further to TORC1's role in coordinating nutrient availability, growth and cell division.
What is the relevance of our observation to mammalian and human aging? Both dietary restriction and inhibition of TOR activity have been linked to lifespan extension in mice 
. At the same time, epidemiological studies in humans have found an association between longevity and long telomeres 
. Because our study demonstrates that dietary restriction and consequent reduction in TOR activity lead to reduction of telomere length, it will be important to determine whether reduced signaling in response to dietary restriction through this highly conserved nutrient and growth related pathway also reduces telomere length in mammals.