Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p

Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.     

The emerging roles of ubiquitin-like protein Urm1 in eukaryotes

The ubiquitin related modifier Urm1 protein was firstly identified in the yeast Saccharomyces cerevisiae, and was later found to play important roles in different eukaryotes. By the assistance of an E1-like activation enzyme Uba4, Urm1 can function as a modifier to target proteins, called urmylation. The thioredoxin peroxidase Ahp1 was the only identified Urm1 target in the early time. Recently, many other Urm1 targets were identified, which is important for us to fully understand functions of urmylation. Urm1 can also function as a sulfur carrier to play a key role in tRNAs thiolation. Mechanisms of the Urm1 in protein and RNA modifications were finely revealed in the past few years. Biological and physiological functions of Urm1 were also found in different organisms. In this review, we will summarize these emerging progresses.     

The dual role of ubiquitin-like protein Urm1 as a protein modifier and sulfur carrier

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.     

A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes

Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin related modifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Deltaurm1 and Deltauba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.     

Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1

We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three-dimensional structure of a ubiquitin-related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin-like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%-26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin-related proteins evolved from a protein-based sulfide donor system of the molybdopterin synthase type.     

Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm⁵s²U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm⁵s²U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.     

Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p

The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance.     

TOR regulation of AGC kinases in yeast and mammals

The TOR (target of rapamycin), an atypical protein kinase, is evolutionarily conserved from yeast to man. Pharmacological studies using rapamycin to inhibit TOR and yeast genetic studies have provided key insights on the function of TOR in growth regulation. One of the first bona fide cellular targets of TOR was the mammalian protein kinase p70 S6K (p70 S6 kinase), a member of a family of kinases called AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases, which include PKA (cAMP-dependent protein kinase A), PKG (cGMP-dependent kinase) and PKC (protein kinase C). AGC kinases are also highly conserved and play a myriad of roles in cellular growth, proliferation and survival. The AGC kinases are regulated by a common scheme that involves phosphorylation of the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1), and phosphorylation at one or more sites at the C-terminal tail. The identification of two distinct TOR protein complexes, TORC1 (TOR complex 1) and TORC2, with different sensitivities to rapamycin, revealed that TOR, as part of either complex, can mediate phosphorylation at the C-terminal tail for optimal activation of a number of AGC kinases. Together, these studies elucidated that a fundamental function of TOR conserved throughout evolution may be to balance growth versus survival signals by regulating AGC kinases in response to nutrients and environmental conditions. This present review highlights this emerging function of TOR that is conserved from budding and fission yeast to mammals.     

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> FKBP12 binds <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> TOR and its expression in plants leads to rapamycin susceptibility

Background  

The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants.     

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.     

TOR Signaling in Fission Yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.     

TOR signaling in fission yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.     

Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.     

Thinking globally and acting locally with TOR

The target of rapamycin (TOR) pathway regulates ribosome biogenesis, protein synthesis, nutrient import, autophagy and cell cycle progression. After 30 years of concentrated attention, how TOR controls these processes is only now beginning to be understood. Recent advances have identified a wide array of TOR inputs, including amino acids, oxygen, ATP and growth factors, as well the regulatory proteins that facilitate their effects on TOR. Such proteins include AMPK, Rheb and the tumor suppressors LKB1, p53, and Tsc1/2. It has only recently been appreciated that TOR resides in two distinct signaling complexes with differing regulatory roles, only one of which is rapamycin-sensitive, thus opening a new avenue of inquiry into TOR function. Finally, TOR appears to regulate feeding behavior by facilitating communication between organ systems, and is thus implicated in the regulation of glucose and fat homeostasis, and possibly diabetes and obesity. TOR thus functions to coordinate growth-permitting inputs with growth-promoting outputs on both a cellular and an organismal level.     

Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton

The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.     

TOR-autophagy signaling in adult zebrafish models of cardiomyopathy

The target of rapamycin (TOR) kinase is part of an evolutionarily conserved signaling pathway that coordinates cell growth, survival, and autophagy. Previously, pharmacological studies using rapamycin have suggested a cardioprotective effect of TOR signaling inhibition on cardiomyopathy. We found that rapamycin exerts a conserved cardioprotective effect in two adult zebrafish models of cardiomyopathy of different etiology, and provided the first genetic evidence to support a long-term cardioprotective effect of TOR signaling inhibition. Moreover, we detected dynamic TOR-autophagy activities along different stages of cardiomyopathy. This needs to be considered when developing TOR-autophagy-based therapeutics for cardiomyopathy.