Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant AI47389)United States. Dept. of Defense (W81XWH-07-0448)W. M. Keck FoundationJane Coffin Childs Memorial Fund for Medical ResearchLAM Foundation (Fellowship

Hypoxia extends lifespan and neurological function in a mouse model of aging.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging

Reduced dosage of the chromosome axis factor Red1 selectively disrupts the meiotic recombination checkpoint in <i>Saccharomyces cerevisiae</i>

<div><p>Meiotic chromosomes assemble characteristic “axial element” structures that are essential for fertility and provide the chromosomal context for meiotic recombination, synapsis and checkpoint signaling. Whether these meiotic processes are equally dependent on axial element integrity has remained unclear. Here, we investigated this question in <i>S</i>. <i>cerevisiae</i> using the putative condensin allele <i>ycs4S</i>. We show that the severe axial element assembly defects of this allele are explained by a linked mutation in the promoter of the major axial element gene <i>RED1</i> that reduces Red1 protein levels to 20–25% of wild type. Intriguingly, the Red1 levels of <i>ycs4S</i> mutants support meiotic processes linked to axis integrity, including DNA double-strand break formation and deposition of the synapsis protein Zip1, at levels that permit 70% gamete survival. By contrast, the ability to elicit a meiotic checkpoint arrest is completely eliminated. This selective loss of checkpoint function is supported by a <i>RED1</i> dosage series and is associated with the loss of most of the cytologically detectable Red1 from the axial element. Our results indicate separable roles for Red1 in building the structural axis of meiotic chromosomes and mounting a sustained recombination checkpoint response.</p></div

Checkpoint arrest and spore viability require different amounts of Red1 protein.

<p><b>(A)</b> Semiquantitative western blot of a Red1 dosage series. Red1 levels of 2h samples from <i>red1</i>_<i>ycs4S</i><i>/RED1</i> (<i>ycs4S/+</i>, H8218), <i>red1Δ/RED1</i> (H8220, <i>Δ/+</i>), <i>red1</i>_<i>ycs4S</i><i>/red1</i>_<i>ycs4S</i> (<i>ycs4S/ycs4S</i>, H7011), and <i>red1</i>_<i>ycs4S</i> <i>/red1Δ</i> (<i>ycs4S/Δ</i>, H8219) were compared to a 2-fold serial dilution of a whole-cell extract from wild type (<i>+/+</i>, H7797) from the same time course. Fpr3 was used as loading control. <b>(B)</b> Quantitative measurement of total Red1 protein levels in the dosage series relative to Nsp1 at 2h and 3h, n = 3 per time point. Error bars: S.E.M. *: p-value < 0.05, **: < 0.005, paired Student <i>t</i>-test. <b>(C)</b> Ratio of upper (modified) band to lower (unmodified) band correlates with Red1 protein levels. Red1 levels were normalized against Nsp1. Quantifications come from the gels used in the creation of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006928#pgen.1006928.g001" target="_blank">Fig 1B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006928#pgen.1006928.g006" target="_blank">Fig 6B</a>. Linear regression of the data is shown along with adjusted R². Examples of two points are shown in the inset. <b>(D)</b> Spindle pole separation of <i>dmc1Δ rad51Δ</i> (closed circle, H7076), <i>dmc1Δ rad51Δ red1</i>_<i>ycs4S</i><i>/RED1</i> (open square, H8467), <i>dmc1Δ rad51Δ red1Δ/RED1</i> (open upward-facing triangle, H8494), <i>dmc1Δ rad51Δ red1</i>_<i>ycs4S</i><i>/red1</i>_<i>ycs4S</i> (closed squared, H7088), <i>dmc1Δ rad51Δ red1</i>_<i>ycs4S</i><i>/red1Δ</i> (open downward-facing triangle, H8504), and <i>dmc1Δ rad51Δ red1Δ/red1Δ</i> (closed upward-facing triangle, H6023), n = 200. <b>(E)</b> Spore viability of the Red1 dosage series and <i>red1Δ/red1Δ</i> (H8098), n>100. *: p-value < 0.05, **: < 0.005, ***: < e^-5 Student <i>t</i>-test. Samples range in color from dark purple to pale orange as they transition from highest Red1 levels to lowest.</p

<i>ycs4S</i> mutants have decreased levels of Red1.

<p><b>(A)</b> Red1 and Hop1 protein levels of whole-cell extracts from wild type (H7797) and <i>ycs4S</i> (H7011) as determined by western blotting at the indicated time points after meiotic induction. Arrows indicate the two Red1 bands. Bracket indicates positions of phosphorylated Hop1. Nsp1 was used as loading control. <b>(B)</b> Fluorescence-based quantitative measurement of total Red1 protein levels in wild type and <i>ycs4S</i> relative to Nsp1 at 2h, 3h, and 4h, n = 3. Error bars: S.E.M. *: p-value < 0.05, **: p-value < 0.005, paired Student <i>t</i>-test. <b>(C)</b> Western analysis of Red1 and Hop1 protein levels in wild-type, <i>ycg1-2</i> (H8632), <i>ycs4S</i>, and <i>ycs4-2</i> (H8601) strains. Nsp1 was used as loading control. <i>ycg1-2</i> and <i>ycs4-2</i> cultures were shifted to 34°C 1 hour after meiotic induction. <b>(D)</b> <i>RED1</i>, <i>HOP1</i>, <i>REC8</i>, and <i>ZIP1</i> mRNA levels at 2h were measured by RT-qPCR from both wild-type (grey) and <i>ycs4S</i> (cyan) extracts, and normalized against <i>ACT1</i>, n = 3. Error bars: S.D. *: p-value < 0.05, Student <i>t</i>-test.</p

<i>ycs4S</i> mutants have a mutation in the promoter of <i>RED1</i>.

<p><b>(A)</b> Red1 and Hop1 protein levels in wild type (H7797), <i>ycs4S</i> (H7011), <i>pHOP1-RED1</i> (H8849), and <i>pHOP1-RED1</i>_<i>ycs4S</i> (H8850) strains. Phosphorylated Hop1 indicated by bracket. Fpr3 was used as loading control. <b>(B)</b> Spore viability of wild-type, <i>ycs4S</i>, <i>pHOP1-RED1</i>, <i>pHOP1-RED1</i>_<i>ycs4S</i>, and <i>YCS4-13xMYC</i> (H9077) strains, as well as <i>red1-pG162A</i> (H9048) mutant strain and a matched control carrying only a marker insertion at position +400 upstream of the <i>RED1</i> ORF (H9049), n>100. Error bars: S.D. **: p-value: < 0.005, Student <i>t</i>-test. (<b>C</b>) Schematic of the introgressed region in the <i>ycs4S</i> mutant. The genetic background of three separate lines was determined across a 35kb region of chromosome XII, which includes the <i>RED1</i> and <i>YCS4</i> genes. This was compared to a wild type SK1 background. Regions were color-coded as SK1 (blue), YPH499 (yellow), S288C (green), and indeterminate (grey). Positions of the <i>12xMYC</i> tag and the <i>G162A</i> SNP in the promoter of <i>RED1</i> are indicated. <b>(D)</b> Red1 and Hop1 protein levels in wild type and a <i>ycs4S</i> mutant, as well as a <i>red1-pG162A</i> mutant strain and its matched control. Fpr3 was used as loading control.</p

Discovery of 1-(4-(4-Propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a Highly Potent, Selective Mammalian Target of Rapamycin (mTOR) Inhibitor for the Treatment of Cancer

The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1, which was identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC[subscript 50] = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility

Decreased deposition but normal distribution of axis proteins in <i>red1</i>_<i>ycs4S</i> mutants.

<p><b>(A)</b> Red1 binding and <b>(B)</b> Hop1 binding was analyzed on chromosome spreads from wild-type (H7797) and <i>red1</i>_<i>ycs4S</i> (H7011) strains, n = 100 per time point. Patterns were classified into four categories: “No foci”, “Few foci” (less than 20 detectable by eye over background), “Many foci”, or “Short Tracks” (indicative of chromosome axes). Scale bars are 5μm. <b>(C)</b> Hop1 intensity values of nuclei spread at 2h after meiotic induction. Focus intensity across an entire spread was normalized against background intensity of an area of identical size. Average intensity is indicated for wild-type (grey), <i>red1</i>_<i>ycs4S</i> (cyan), <i>rec8Δ</i> (pink, H5187), and <i>red1</i>_<i>ycs4S</i> <i>rec8Δ</i> (orange, H7661) strains, n = 20. Error bars: S.D. ***: p-value < 10⁻⁵, Wilcox test. <b>(D)</b> Red1 chromosomal localization at 3h determined by ChIP-seq in wild-type (black, H119), <i>red1</i>_<i>ycs4S</i> (H7011), <i>rec8Δ</i> (H7660 & H7772), and <i>red1</i>_<i>ycs4S</i> <i>rec8Δ</i> (H7661) strains on chromosomes III and VIII, n = 2. Large black circles indicate the positions of the centromeres. Triangles indicate positions analyzed by ChIP-qPCR in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006928#pgen.1006928.s003" target="_blank">S3B Fig</a>. <b>(E)</b> Percentage of spreads at 2h from time course in <b>(C)</b> exhibiting clumps of Hop1 (arrow in example image), n = 100.</p

<i>red1</i>_<i>ycs4S</i> mutants lack a sustained meiotic DNA damage checkpoint arrest.

<p><b>(A)</b> Sporulation efficiency of <i>red1</i>_<i>ycs4S</i> (H7011), <i>rec8Δ</i> (H5187), and <i>red1</i>_<i>ycs4S</i> <i>rec8Δ</i> (H7661) mutants relative to wild type (H7797), n = 200. <b>(B)</b> Western analysis of phosphorylation levels of Hop1-T318 and H3-T11 from wild-type and <i>red1</i>_<i>ycs4S</i> cells. Fpr3 was used as loading control. <b>(C)</b> Spindle pole separation in fixed cells measured by anti-tubulin immunofluorescence of wild type (black circle), <i>red1</i>_<i>ycs4S</i> (cyan triangle), <i>rec8Δ</i> (pink square), and <i>red1</i>_<i>ycs4S</i> <i>rec8Δ</i> (orange diamond), n = 200. Spindle pole separation is an indication of progression out of meiotic prophase. <b>(D)</b> Spindle pole separation in <i>red1</i>_<i>ycs4S</i> (H7088), <i>rec8Δ</i> (H7161), and <i>red1</i>_<i>ycs4S</i> <i>rec8Δ</i> (H6589) mutants and their control (H7076) in a repair-deficient <i>dmc1Δ rad51Δ</i> background, n = 200. <b>(E)</b> Spindle pole separation of wild type (black closed circle), <i>red1</i>_<i>ycs4S</i> (cyan closed triangle), <i>red1-pG162A</i> (H9080, cyan open triangle) and its matched control (H9078, black open circle) strains in a <i>dmc1Δ rad51Δ</i> background to measure checkpoint activity, n = 200. <b>(F)</b> Spindle pole separation of wild type (black closed circle), <i>red1</i>_<i>ycs4S</i> (cyan closed triangle), <i>pHOP1-RED1</i> (H8851, black open circle), and <i>pHOP1-RED1</i>_<i>ycs4S</i> (H8852, cyan open triangle) strains in a <i>dmc1Δ rad51Δ</i> background to measure checkpoint activity, n = 200.</p

Architecture of the Mitochondrial Calcium Uniporter

Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture and is stabilized by a coiled coil motif protruding in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel