Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase

TS (thymidylate synthase) is a key enzyme in the de novo biosynthesis of dTMP, and is indispensable for DNA replication. Previous studies have shown that intracellular degradation of the human enzyme [hTS (human thymidylate synthase)] is mediated by the 26S proteasome, and occurs in a ubiquitin-independent manner. Degradation of hTS is governed by a degron that is located at the polypeptide''s N-terminus that is capable of promoting the destabilization of heterologous proteins to which it is attached. The hTS degron is bipartite, consisting of two subdomains: an IDR (intrinsically disordered region) that is highly divergent among mammalian species, followed by a conserved amphipathic α-helix (designated hA). In the present report, we have characterized the structure and function of the hTS degron in more detail. We have conducted a bioinformatic analysis of interspecies sequence variation exhibited by the IDR, and find that its hypervariability is not due to diversifying (or positive) selection; rather, it has been subjected to purifying (or negative) selection, although the intensity of such selection is relaxed or weakened compared with that exerted on the rest of the molecule. In addition, we have verified that both subdomains of the hTS degron are required for full activity. Furthermore, their co-operation does not necessitate that they are juxtaposed, but is maintained when they are physically separated. Finally, we have identified a ‘cryptic’ degron at the C-terminus of hTS, which is activated by the N-terminal degron and appears to function only under certain circumstances; its role in TS metabolism is not known.     

Flexible Regions within IκBα Create the Ubiquitin-independent Degradation Signal

Homeostatic regulation of NF-κB requires the continuous synthesis of IκBα and its rapid degradation by the proteasome through a ubiquitin-independent pathway. We previously showed that the ubiquitin-independent degradation signal of unbound IκBα was located in the C-terminal PEST region, and we have now identified a single tyrosine, Tyr-289, and determined that the hydrophobic character of the tyrosine is important for the rapid turnover of IκBα. The sequence composition of the PEST peptide surrounding this Tyr-289 imposes a distinct polyproline II conformation. Enhancing the polyproline II helix formation correlates with slower degradation rates of unbound IκBα. We have further identified a degradation signal located within the 5th ankyrin repeat that is functional once the C terminus is removed. Both the C-terminal and 5th ankyrin repeat degradation signals have inherent flexibility and specific hydrophobic residue(s), which together constitute the ubiquitin-independent degradation signal for IκBα.     

The Proline-rich N-terminal Domain of G18 Exhibits a Novel G Protein Regulatory Function

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Gα_i, whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Gα_i1 and Gα_o. Surprisingly, in the presence of fluoroaluminate (AlF₄⁻) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18ΔC) but not to its GoLoco region (ΔNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Gα_i/o. Neither G18wt nor any G18 mutant affected the GTPase activity of Gα_i1 or Gα_o. In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [³⁵S]GTPγS (guanosine 5′-O-[γ-thio]triphosphate) to Gα_i1, the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [³⁵S]GTPγS binding to Gα_o, albeit to a lesser extent than the effect of the GoLoco region on Gα_i1. Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the G_i/o subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.     

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ^F. Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA₂·SpoIISB₂ heterotetramer. CSpoIISA has a single domain α/β structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.     

The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron

Ornithine decarboxylase (ODC), a homodimeric enzyme with a rate-limiting function in polyamine biosynthesis, is subject to a feedback control involving its selective proteolysis. Targeting of ODC monomers to the proteasome is mediated by ODC antizyme (OAZ), the expression of which is induced by high levels of polyamines. Here, we report our analysis of the N-terminal degron in Saccharomyces cerevisiae ODC and the mechanism of its antizyme-dependent targeting. This ∼ 45-residue domain of ODC [termed ODC degradation signal (ODS)] is essential for degradation of ODC. Extensive mutagenesis indicated that it is not a specific sequence within ODS that is important but, rather, its unstructured nature. Consistent with this conclusion, ODS could be functionally replaced by an unrelated unstructured domain. We show that increasing the distance of ODS to the rest of the ODC protein reduced the dependence on Oaz1 for targeting, indicating that exposure of ODS is critical for its function. Disruption of ODC dimers by introducing interface mutations, in contrast, was insufficient for targeting. Binding of Oaz1 to ODC monomers is thus required to activate ODS. Fusion of ODS to the N terminus of Ura3 was sufficient to convert it into a ubiquitin-independent substrate of the proteasome. By contrast, ODS failed to destabilize maltose-binding protein or dihydrofolate reductase, indicating that this degron only operates in an appropriate structural context that enables rapid unfolding.     

Structure of the Nuclear Factor κB-inducing Kinase (NIK) Kinase Domain Reveals a Constitutively Active Conformation

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5′-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.     

Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)

Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Δypc1Δydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca²⁺ for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2ΔN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2ΔN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2ΔN13, but not ACER2ΔN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2ΔN13 or ACER2ΔN36 inhibited the glycosylation of integrin β1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca²⁺ for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.     

The Maturational Refolding of the β-Hairpin Motif of Equine Infectious Anemia Virus Capsid Protein Extends Its Helix α1 at Capsid Assembly Locus

A retroviral capsid (CA) protein consists of two helical domains, CA^N and CA^C, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr²⁰ to Pro¹⁷. This coil to helix transition involves the conserved sequence of Thr¹⁶-Pro¹⁷-Arg¹⁸ (Ser¹⁶-Pro¹⁷-Arg¹⁸ in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA^N oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg¹⁸, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA^N oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA^N domain oligomerization, independent to the faster EIAV-CA^C domain dimerization.     

Processing of autophagic protein LC3 by the 20S proteasome

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.     

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF486523","term_id":"1121787749","term_text":"EF486523"}}EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.     

The Stability of the Small Nucleolar Ribonucleoprotein (snoRNP) Assembly Protein Pih1 in Saccharomyces cerevisiae Is Modulated by Its C Terminus

Pih1 is an unstable protein and a subunit of the R2TP complex that, in yeast Saccharomyces cerevisiae, also contains the helicases Rvb1, Rvb2, and the Hsp90 cofactor Tah1. Pih1 and the R2TP complex are required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. Purified Pih1 tends to aggregate in vitro. Molecular chaperone Hsp90 and its cochaperone Tah1 are required for the stability of Pih1 in vivo. We had shown earlier that the C terminus of Pih1 destabilizes the protein and that the C terminus of Tah1 binds to the Pih1 C terminus to form a stable complex. Here, we analyzed the secondary structure of the Pih1 C terminus and identified two intrinsically disordered regions and five hydrophobic clusters. Site-directed mutagenesis indicated that one predicted intrinsically disordered region IDR2 is involved in Tah1 binding, and that the C terminus of Pih1 contains multiple destabilization or degron elements. Additionally, the Pih1 N-terminal domain, Pih1^1–230, was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih1^1–230 as well as a shorter Pih1 N-terminal fragment Pih1^1–195 is able to bind Rvb1/Rvb2 heterocomplex. However, the sequence between the two disordered regions in Pih1 significantly enhances the Pih1 N-terminal domain binding to Rvb1/Rvb2. Based on these data, a model of protein-protein interactions within the R2TP complex is proposed.     

Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores

How individual nucleoporins (Nups) perform their role in nuclear pore structure and function is largely unknown. In this study, we examined the structure of purified Nup170 to obtain clues about its function. We show that Nup170 adopts a crescent moon shape with two structurally distinct and separable domains, a β-propeller N terminus and an α-solenoid C terminus. To address the individual roles of each domain, we expressed these domains separately in yeast. Notably, overexpression of the Nup170 C domain was toxic in nup170Δ cells and caused accumulation of several Nups in cytoplasmic foci. Further experiments indicated that the C-terminal domain anchors Nup170 to nuclear pores, whereas the N-terminal domain functions to recruit or retain a subset of Nups, including Nup159, Nup188, and Pom34, at nuclear pores. We conclude that Nup170 performs its role as a structural adapter between cytoplasmically oriented Nups and the nuclear pore membrane.     

Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.     

Crystal structure of the heteromolecular chaperone, AscE-AscG, from the type III secretion system in Aeromonas hydrophila

Background

The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62–116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion.

Methodology/Principal Findings

Here, we report the crystal structure of the ordered AscG_1–61 region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG_1–61 complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4–12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG_1–61 comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG_1–61 complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex.

Conclusion/Significance

The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.     

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single α-helical globular domain, microplusin consists of five α-helices: α1 (residues Gly-9 to Arg-21), α2 (residues Glu-27 to Asn-40), α3 (residues Arg-44 to Thr-54), α4 (residues Leu-57 to Tyr-64), and α5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.     

Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.     

Structural determinants for the intracellular degradation of human thymidylate synthase

Thymidylate synthase (EC 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for DNA replication in actively dividing cells. The enzyme is a critical target at which chemotherapeutic agents such as fluoropyrimidines (e.g., 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and folic acid analogues (e.g., raltitrexed, LY231514, ZD9331, and BW1843U89) are directed. These agents exert their effects through the generation of metabolites that bind the active site of TS and inhibit catalytic activity. The binding of ligands to the TS molecule leads to dramatic changes in the conformation of the enzyme, particularly within the C-terminal domain. Stabilization of the enzyme and an increase in its intracellular level are associated with ligand binding and may be important in cellular response to TS-directed drugs. In the present study, we have examined molecular features of the TS molecule that control its degradation. We find that the C-terminal conformational shift is not required for ligand-mediated stabilization of the enzyme. In addition, we demonstrate that the N-terminus of the TS polypeptide, which is extended in the mammalian enzyme and is disordered in crystal structures, is a primary determinant of the enzyme's half-life. Finally, we show that TS turnover is carried out by the 26S proteasome in a ubiquitin-independent manner. These findings provide the basis for a mechanistic understanding of TS degradation and its regulation by antimetabolites.