Ubc6p and ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase.
The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T(4) deiodination to T(3). Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T(4). 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 micro M T(4) caused rapid loss of CysD2 (t(1/2) = approximately 30 min). Clasto-lactacystin beta-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T(4). 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.
Thyroid Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Diego
D
Botero
Balazs
B
Gereben
Carla
C
Goncalves
Lucia A
LA
De Jesus
John W
JW
Harney
Antonio C
AC
Bianco
D015153:Blotting, Western; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D017353:Gene Deletion; D006207:Half-Life; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000097564:Iodothyronine Deiodinase Type II
D003546:Cysteine Endopeptidases; D005690:Galactose; D007453:Iodide Peroxidase; D009097:Multienzyme Complexes; D046988:Proteasome Endopeptidase Complex; D029701:Saccharomyces cerevisiae Proteins; D013974:Thyroxine; D014284:Triiodothyronine; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D000097564:Iodothyronine Deiodinase Type II; C066752:UBC1 protein, S cerevisiae; C082461:UBC6 protein, S cerevisiae; C471617:UBE2G2 protein, human; C489967:UBE2J1 protein, human