Human thymidylate synthase (hTS; EC 2.1.1.45) is one of a small group of proteasomal substrates whose intracellular degradation occurs in a ubiquitin-independent manner. Previous studies have shown that proteolytic breakdown of the hTS polypeptide is directed by an intrinsically disordered 27-residue domain at the N-terminal end of the molecule. This domain, in co-operation with an α-helix spanning amino acids 31–45, functions as a degron, in that it has the ability to destabilize a heterologous polypeptide to which it is attached. In the present study, we provide evidence indicating that it is the 26S isoform of the proteasome that is responsible for intracellular degradation of the hTS polypeptide. In addition, we have used targeted in vitro mutagenesis to show that an Arg–Arg motif at residues 10–11 is required for proteolysis, an observation that was confirmed by functional analysis of the TS N-terminus from other mammalian species. The effects of stabilizing mutations on hTS degradation are maintained when the enzyme is provided with an alternative means of proteasome association; thus such mutations perturb one or more post-docking steps in the degradation pathway. Surprisingly, deletion mutants missing large segments of the disordered domain still function as proteasomal substrates; however, degradation of such mutants occurs by a mechanism that is distinct from that for the wild-type protein. Taken together, our results provide information on the roles of specific subregions within the intrinsically disordered N-terminal domain of hTS in regulation of degradation, leading to a deeper understanding of mechanisms underlying the ubiquitin-independent proteasomal degradation pathway.
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Research Article|
October 25 2010
Functional dissection of the N-terminal degron of human thymidylate synthase
Sandra P. Melo;
Sandra P. Melo
1
1Department of Biological Sciences and Center for Colon Cancer Research, University of South Carolina, Columbia, SC 29208, U.S.A.
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Asami Yoshida;
Asami Yoshida
2
1Department of Biological Sciences and Center for Colon Cancer Research, University of South Carolina, Columbia, SC 29208, U.S.A.
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Franklin G. Berger
Franklin G. Berger
3
1Department of Biological Sciences and Center for Colon Cancer Research, University of South Carolina, Columbia, SC 29208, U.S.A.
3To whom correspondence should be addressed (email fgberger@mailbox.sc.edu).
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Publisher: Portland Press Ltd
Received:
July 09 2010
Revision Received:
August 30 2010
Accepted:
September 03 2010
Accepted Manuscript online:
September 03 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 432 (1): 217–226.
Article history
Received:
July 09 2010
Revision Received:
August 30 2010
Accepted:
September 03 2010
Accepted Manuscript online:
September 03 2010
Citation
Sandra P. Melo, Asami Yoshida, Franklin G. Berger; Functional dissection of the N-terminal degron of human thymidylate synthase. Biochem J 15 November 2010; 432 (1): 217–226. doi: https://doi.org/10.1042/BJ20101027
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