Thioredoxin-related Protein 32 is an arsenite-regulated Thiol Reductase of the proteasome 19 S particle.
Perturbation of the cytoplasmic protein folding environment by exposure to oxidative stress-inducing As(III)-containing compounds challenges the ubiquitin-proteasome system. Here we report on mass spectrometric analysis of As(III)-induced changes in the proteasome's composition in samples prepared by stable isotope labeling with amino acids in cell culture, using mammalian cells in which TRP32 (thioredoxin-related protein of 32 kDa; also referred to as TXNL1) was identified as a novel subunit of the 26 S proteasome. Quantitative genetic interaction mapping, using the epistatic miniarray profiling approach, identified a functional connection between TRP32 and the proteasome. Deletion of txl1, the Schizosaccharomyces pombe homolog of TRP32, results in a slow growth phenotype when combined with deletion of cut8, a gene required for normal proteasome localization. Deletion analysis in vivo, chemical cross-linking, and manipulation of the ATP concentration in vitro during proteasome immunopurification revealed that the C-terminal domain of mammalian TRP32 binds the 19 S regulatory particle in proximity to the proteasome substrate binding site. Thiol modification with polyethylene glycol-maleimide showed disulfide bond formation at the active site of TRP32 in cells exposed to As(III). Pulse-chase labeling showed that TRP32 is a stable protein whose half-life of >6 h is surprisingly reduced to 1 h upon exposure of cells to As(III). These findings reveal a previously undescribed thiol reductase at the proteasome's regulatory particle.
Kimmel Center for Biology and Medicine of the Skirball Institute , New York University School of Medicine, New York, New York 10016, USA.
R Luke
RL
Wiseman
King-Tung
KT
Chin
Cole M
CM
Haynes
Ariel
A
Stanhill
Chong-Feng
CF
Xu
Assen
A
Roguev
Nevan J
NJ
Krogan
Thomas A
TA
Neubert
David
D
Ron
D000818:Animals; D018053:Arsenites / Q000633:toxicity*; D002460:Cell Line; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D051379:Mice; D016254:Mutagenesis, Insertional / Q000187:drug effects; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D018053:Arsenites; D004220:Disulfides; C504766:TXNL1 protein, human; C504767:Txnl1 protein, mouse; D013879:Thioredoxins; D011490:Protein Disulfide Reductase (Glutathione); D046988:Proteasome Endopeptidase Complex; C119011:26S proteasome non-ATPase regulatory subunit 13; C015001:arsenite