TOR signalling and control of cell growth.

TOR, phosphatidylinositol 3-kinase, p70s6k, and 4E-BP1 have recently emerged as components of a major signalling pathway that is dedicated to protein translation and thus to cell growth. This pathway appears to be conserved, at least in part, in yeast, slime molds, plants, flies, and mammals. TOR and phosphatidylinositol 3-kinase control p70s6k and 4E-BP1, which, in turn, directly control the translation initiation machinery.

Evaluation of the potential use of adipose-derived mesenchymal stromal cells in the treatment of canine atopic dermatitis: a pilot study.

Stem cells and their potential therapeutic uses in human and veterinary medicine have generated considerable interest. These cells have a number of potentially unique immunologic properties; most notable are their reported regenerative and antiinflammatory capabilities. The aim of this prospective pilot study was to evaluate the efficacy of intravenously administered autogenous adipose-derived mesenchymal stem cells (AD-MSCs) in the treatment of canine atopic dermatitis. AD-MSCs administered intravenously at a dose of 1.3 million cells/kg did not significantly reduce the clinical signs of canine atopic dermatitis or the owner-assessed pruritus level.

Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast.

In Saccharomyces cerevisiae, amino acid permeases are divided into two classes. One class, represented by the general amino acid permease GAP1, contains permeases regulated in response to the nitrogen source. The other class, including the high affinity tryptophan permease, TAT2, consists of the so-called constitutive permeases. We show that TAT2 is regulated at the level of protein stability. In exponentially growing cells, TAT2 is in the plasma membrane and also accumulates in internal compartments of the secretory pathway. Upon nutrient deprivation or rapamycin treatment, TAT2 is transported to and degraded in the vacuole. The ubiquitination machinery and lysine residues within the NH(2)-terminal 31 amino acids of TAT2 mediate ubiquitination and degradation of the permease. Starvation-induced degradation of internal TAT2 is blocked in sec18, sec23, pep12, and vps27 mutants, but not in sec4, end4, and apg1 mutants, suggesting that, upon nutrient limitation, internal TAT2 is diverted from the late secretory pathway to the vacuolar pathway. Furthermore, our results suggest that TAT2 stability and sorting are controlled by the TOR signaling pathway, and regulated inversely to that of GAP1.

Cell wall stress depolarizes cell growth via hyperactivation of RHO1.

Cells sense and physiologically respond to environmental stress via signaling pathways. Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton. We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage. The redistribution of FKS1 is dependent on the actin cytoskeleton. Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch. WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells. The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1. Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

A mechanism of protein localization: the signal hypothesis and bacteria.

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.

Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.

FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle.

Homeo domain of the yeast repressor alpha 2 is a sequence-specific DNA-binding domain but is not sufficient for repression.

The alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of cell type in the yeast Saccharomyces cerevisiae. It represses transcription of a group of cell type-specific genes by binding to an operator located upstream of each target gene. Fifteen in-frame deletions within the coding region of the MAT alpha 2 gene were constructed. The deletion alleles were examined for phenotypes conferred in vivo, and the encoded mutant proteins were assayed for ability to bind specifically to the operator in vitro. This analysis has revealed that the sequence-specific DNA-binding domain of alpha 2 is located within a region of 68 amino acids. This region of alpha 2 has significant homology with the homeo domain, a conserved sequence found in the products of several Drosophila homeotic and segmentation genes. In addition, there is a class of mutant alpha 2 proteins that binds tightly and specifically to the operator in vitro, but fails to repress transcription in vivo.

Cyclosporin A, FK506 and rapamycin: more than just immunosuppression.

The mechanisms of action of the immunosuppressive drugs cyclosporin A (CsA), FK506 and rapamycin are strikingly conserved from yeast to human T cells. Recent results obtained with yeast corroborate calcineurin as the target of CsA-cyclophilin and FK506-FKBP complexes, and reveal a phosphatidylinositol 3-kinase homologue as the target of the rapamycin-FKBP complex.

mTOR-what does it do?

Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. TOR, which was originally discovered in yeast, is conserved in all eukaryotes including plants, worms, flies, and mammals. The discovery of TOR led to a fundamental change in how we think about cell growth. It is not a spontaneous process that just happens when building blocks (nutrients) are available, but rather a highly regulated, plastic process controlled by TOR-dependent signaling pathways. TOR is found in 2 structurally and functionally distinct multiprotein complexes, TORC1 and TORC2. The 2 TOR complexes, like TOR itself, are highly conserved. Mammalian TORC1 (mTORC1) is rapamycin sensitive and contains mTOR, raptor, and mLST8. TORC1 in yeast and mammals mediates temporal control of cell growth by regulating several cellular processes, including translation, transcription, ribosome biogenesis, nutrient transport, and autophagy. mTORC2 is rapamycin insensitive and contains mTOR, rictor, mSIN1, PRR5, and mLST8. TORC2 in yeast and mammals mediates spatial control of cell growth by regulating the actin cytoskeleton. Thus, the 2 TOR complexes constitute an ancestral signaling network conserved throughout eukaryotic evolution to control the fundamental process of cell growth. As a central controller of cell growth, TOR plays a key role in development and aging and has been implicated in disorders such as cancer, cardiovascular disease, obesity, and diabetes. The challenge now is to understand the role of mTOR signaling to coordinate and integrate overall body growth in multicellular organisms.

The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton.

In Saccharomyces cerevisiae, the phosphatidylinositol kinase homologue Tor2 controls the cell-cycle-dependent organisation of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2 [1,2]. Four Rho1 effectors are known, protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signalling protein Skn7 [2,3]. Rho1 has been suggested to signal to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organisation, however [2,4]. We have further investigated the role of Rho1 in controlling actin organisation and have analysed which of the Rho1 effectors mediates Tor2 signalling to the actin cytoskeleton. We show that some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganised actin cytoskeleton. Both the growth defect and the actin organisation defect of the rho1-2ts mutant were suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescued a tor2ts mutant, and deletion of BNI1 or SKN7 did not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppressed the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade.

The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S. cerevisiae.

Polyphosphoinositides have many roles in cell signalling and vesicle trafficking [1-3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast. PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro. A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transferase (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro. Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

Two FK506 resistance-conferring genes in Saccharomyces cerevisiae, TAT1 and TAT2, encode amino acid permeases mediating tyrosine and tryptophan uptake.

The macrocyclic lactone FK506 exerts immunosuppressive effects on T lymphocytes by interfering with signal transduction leading to T-cell activation and also inhibits the growth of eukaryotic microorganisms, including Saccharomyces cerevisiae. We reported previously that an FK506-sensitive target in S. cerevisiae is required for amino acid import and that overexpression of two new genes, TAT1 and TAT2 (formerly called TAP1 and TAP2), confers resistance to the drug. Here we report that TAT1 and TAT2 encode novel members of the yeast amino acid permease family composed of integral membrane proteins that share 30 to 40% identity. TAT1 is the tyrosine high-affinity transporter, which also mediates low-affinity or low-capacity uptake of tryptophan. TAT2 is the tryptophan high-affinity transporter. FK506 does not reduce the levels of TAT1 and TAT2 transcripts, indicating that the inhibition of amino acid transport by the drug is posttranscriptional.

PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast.

PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae.

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.

TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TOR1-1 and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycin-sensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.

TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine.

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.

Regional bivalent-univalent pairing versus trivalent pairing of a trisomic chromosome in Saccharomyces cerevisiae.

In meiosis I, homologous chromosomes pair, recombine and segregate to opposite poles. These events and subsequent meiosis II ensure that each of the four meiotic products has one complete set of chromosomes. In this study, the meiotic pairing and segregation of a trisomic chromosome in a diploid (2n + 1) yeast strain was examined. We find that trivalent pairing and segregation is the favored arrangement. However, insertions near the centromere in one of the trisomic chromosomes leads to preferential pairing and segregation of the "like" centromeres of the remaining two chromosomes, suggesting that bivalent-univalent pairing and segregation is favored for this region.

TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.