The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

Maria Marjorette O Peña,Sandra P Melo,Yang-Yang Xing,Kenneth White,Karen W Barbour,Franklin G Berger

doi:10.1074/jbc.m109.038455

Maria Marjorette O Peña, Sandra P Melo + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.038455

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Abstract

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 alpha-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(alpha)-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.

Highlights

Thine decarboxylase (ODC)3 (4 – 6), c-Fos [7, 8], p21Cip1 [9, 10], hepatitis virus F protein [11], and c-Jun [12], among others
To determine whether the region can act as a degron, i.e. if it is capable of destabilizing a completely unrelated protein, we tested its effect on the intracellular degradation of enhanced green fluorescence protein (eGFP), a protein that normally has a very long halflife
A chimeric polypeptide in which the first 30 residues of thymidylate synthase (TS) were fused to the N-terminal end of the eGFP reporter exhibited slight instability compared with eGFP (Fig. 1A), indicating that the disordered region has little if any impact on the stability of the reporter

Summary

EXPERIMENTAL PROCEDURES

Cells and Cell Culture—All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. The strips were equilibrated for 15 min in a buffer containing 6 M urea, 75 mM Tris-HCl, pH 8.8, 29.3% glycerol, 2% SDS, 10 mg/ml dithiothreitol, and 0.002% bromphenol blue followed by a further treatment for 15 min in a similar buffer but containing 25 mg/ml iodoacetamide instead of dithiothreitol. They were transferred directly onto 12.5% pre-cast SDS-PAGE gels and kept in place with 0.5% agarose. Disorder propensities were calculated using PONDR௡ VL-XT software [21,22,23]

RESULTS

Penultimate residue

DISCUSSION