The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway.
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.
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA.
Maria Marjorette O
MM
Peña
Sandra P
SP
Melo
Yang-Yang
YY
Xing
Kenneth
K
White
Karen W
KW
Barbour
Franklin G
FG
Berger
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D008168:Lung / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D025801:Ubiquitin; D013940:Thymidylate Synthase; D046988:Proteasome Endopeptidase Complex