The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an ␣-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N ␣ -acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.Regulated protein degradation within the cell is carried out primarily by the 26 S proteasome, a large 2-MDa complex consisting of several dozen proteins that function in recognizing and degrading its target substrates (1, 2). Typically, covalent attachment of polyubiquitin chains serves as the primary signal for target recognition by the proteasome (1, 2). However, in recent years several proteasomal substrates have been shown to be degraded without a requirement for ubiquitin modification (for a recent review, see Ref.3). Such substrates include ornithine decarboxylase (ODC) 3 (4 -6), c-Fos (7, 8), p21 Cip1 (9, 10), hepatitis virus F protein (11), and c-Jun (12), among others. Although the number of substrates identified as degraded by a ubiquitin-independent mechanism remains small, recent biochemical analyses indicate that the process may be more widespread than previously thought and contributes significantly to the regulation of protein turnover (13).Among the known substrates of the ubiquitin-independent degradation pathway, ODC has been the most studied. The degradation signal for ODC is composed of a disordered, flexible domain formed by a 37-residue region at the C terminus (4 -6). The region mediates docking of the ODC polypeptide to the proteasome and initiates its entry into the proteasomal chamber, where proteolysis proceeds in a C-to N-terminal direction (4 -6). This process is stimulated by an accessory protein termed antizyme, which binds ODC and increases the availability of i...
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.
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