Ubc6p and Ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase

Diego Botero, Balazs Gereben, Carla Goncalves, Lucia A. De Jesus, John W. Harney, Antonio C. Bianco

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T4 deiodination to T3. Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T4. To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter. CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells. Treatment with 100 mg/ml cycloheximide or 30 μM T4 caused rapid loss of CysD2 (t1/2 = ∼30 min). Clasto-lactacystin β-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T4. Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2.

Original languageEnglish
Pages (from-to)1999-2007
Number of pages9
JournalMolecular Endocrinology
Volume16
Issue number9
DOIs
Publication statusPublished - Sep 1 2002

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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