Terunao Takahara

Transient Sequestration of TORC1 into Stress Granules during Heat Stress

The target of rapamycin complex 1 (TORC1) is a central kinase that coordinates nutrient availability with eukaryotic cell growth. Although TORC1 signaling is repressed by various stresses in yeast, the underlying mechanisms remain elusive. Here we report that TORC1 signaling upon heat stress is regulated by stress granules (SGs), which are cytoplasmic foci formed under certain stresses. Ectopic formation of SGs achieved by Pbp1 overexpression in unstressed cells sequesters TORC1 in this compartment, thereby blunting TORC1 signaling. Upon heat stress, a physiological SG-inducing condition, TORC1 is also recruited to SGs, which delays reactivation of TORC1 signaling during recovery from heat stress. Moreover, TORC1 reactivation is directed through SG disassembly, suggesting that SGs act as a key determinant for TORC1 reactivation during recovery from heat stress. Furthermore, this mechanism contributes to reduction of heat-induced mutations. Thus, TORC1 signaling is coupled to heat-induced SGs to protect cells from DNA damage.

Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region

The mammalian target of rapamycin (mTOR) plays a pivotal role in the regulation of cell growth in response to a variety of signals such as nutrients and growth factors. mTOR forms two distinct complexes in vivo. mTORC1 (mTOR complex 1) is rapamycin-sensitive and regulates the rate of protein synthesis in part by phosphorylating two well established effectors, S6K1 (p70 ribosomal S6 kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). mTORC2 is rapamycin-insensitive and likely regulates actin organization and activates Akt/protein kinase B. Here, we show that mTOR forms a multimer via its N-terminal HEAT repeat region in mammalian cells. mTOR multimerization is promoted by amino acid sufficiency, although the state of multimerization does not directly correlate with the phosphorylation state of S6K1. mTOR multimerization was insensitive to rapamycin treatment but hindered by butanol treatment, which inhibits phosphatidic acid production by phospholipase D. We also found that mTOR forms a multimer in both mTORC1 and mTORC2. In addition, Saccharomyces cerevisiae TOR proteins Tor1p and Tor2p also exist as homomultimers. These results suggest that TOR multimerization is a conserved mechanism for TOR functioning.

Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2

ABSTRACT Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2.

Isolation of Hyperactive Mutants of Mammalian Target of Rapamycin

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growth-related processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1 (mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from loss-of-function analyses using rapamycin and (molecular)genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.

Sphingolipids Regulate the Yeast High-Osmolarity Glycerol Response Pathway

ABSTRACT The yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.

TORC1 of fission yeast is rapamycin-sensitive

The target of rapamycin (TOR) protein kinase plays central roles in the regulation of cell growth in response to nutritional availability. TOR forms two distinct multiprotein complexes termed TOR complex 1 (TORC1) and TORC2. Typically, only the activity of TORC1 is inhibited by the immunosuppressant rapamycin. Although rapamycin strongly inhibits cell growth of the budding yeast Saccharomyces cerevisiae through inhibition of TORC1, growth of the fission yeast Schizosaccharomyces pombe appears to be resistant to rapamycin. Here, we demonstrate that rapamycin inhibits the kinase activity of S. pombe TORC1 in vitro in a similar manner to TORC1 of other organisms. We furthermore show that incomplete inhibition of TORC1 by rapamycin underlies the apparent rapamycin resistance of S. pombe. In the presence of caffeine, which potentially lowers TORC1 activity, the growth of wild‐type S. pombe cells is sensitive to rapamycin in a TORC1‐dependent manner. Moreover, treatment of S. pombe cells with rapamycin plus caffeine induces starvation‐specific gene expression and autophagy, similarly to cells with reduced TORC1 activity. These results indicate that rapamycin does inhibit TORC1 in S. pombe, but the inhibition is not sufficient to cause a growth defect. These findings establish a universal action of rapamycin on TORC1 inhibition.

Evolutionarily conserved regulation of TOR signalling.

The target of rapamycin (TOR) is an evolutionarily conserved protein kinase that regulates cell growth in response to various environmental as well as intracellular cues through the formation of 2 distinct TOR complexes (TORC), TORC1 and TORC2. Dysregulation of TORC1 and TORC2 activity is closely associated with various diseases, including diabetes, cancer and neurodegenerative disorders. Over the past few years, new regulatory mechanisms of TORC1 and TORC2 activity have been elucidated. Furthermore, recent advances in the study of TOR inhibitors have revealed previously unrecognized cellular functions of TORC1. In this review, we briefly summarize the current understanding of the evolutionarily conserved TOR signalling from upstream regulators to downstream events.

Changes in cell morphology are coordinated with cell growth through the TORC1 pathway.

BACKGROUND Growth rate is determined not only by extracellular cues such as nutrient availability but also by intracellular processes. Changes in cell morphology in budding yeast, mediated by polarization of the actin cytoskeleton, have been shown to reduce cell growth. RESULTS Here we demonstrate that polarization of the actin cytoskeleton inhibits the highly conserved Target of Rapamycin Complex 1 (TORC1) pathway. This downregulation is suppressed by inactivation of the TORC1 pathway regulatory Iml1 complex, which also regulates TORC1 during nitrogen starvation. We further demonstrate that attenuation of growth is important for cell recovery after conditions of prolonged polarized growth. CONCLUSIONS Our results indicate that extended periods of polarized growth inhibit protein synthesis, mass accumulation, and the increase in cell size at least in part through inhibiting the TORC1 pathway. We speculate that this mechanism serves to coordinate the ability of cells to increase in size with their biosynthetic capacity.

Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.

Nuclear ALG-2 protein interacts with Ca2+ homeostasis endoplasmic reticulum protein (CHERP) Ca2+-dependently and participates in regulation of alternative splicing of inositol trisphosphate receptor type 1 (IP3R1) pre-mRNA.

Background: ALG-2 is present both in the cytoplasm and nucleus, but little is known about its nuclear function. Results: ALG-2 interacts with the SR superfamily protein CHERP and accumulates at nuclear speckles in a Ca2+-dependent manner. Conclusion: ALG-2 and CHERP participate in alternative splicing. Significance: We propose a new role of ALG-2 and CHERP in post-transcriptional processing in the nucleus. The intracellular Ca2+ signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca2+-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca2+-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca2+ homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca2+ mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca2+-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca2+ mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40–42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca2+ signaling pathways.