Regulation of the Proapoptotic ARTS Protein by Ubiquitin-mediatedDegradation

Rona Lotan,Asaf Rotem,Hedva Gonen,John P.M Finberg,Stav Kemeny,Hermann Steller,Aaron Ciechanover,Sarit Larisch

doi:10.1074/jbc.m501955200

Abstract

ARTS is a mitochondrial protein that promotes apoptosis induced by a variety of proapoptotic stimulators. ARTS induces apoptosis, at least in part, through binding to and antagonizing IAPs (inhibitors of apoptosis proteins). As a result of ARTS binding to IAPs, caspase inhibition is removed and apoptosis can be executed. Here we show that high cellular levels of ARTS protein sensitize cells toward apoptosis. Accordingly, in healthy cells ARTS levels are kept low through constant ubiquitin-mediated degradation. Upon proapoptotic stimuli, the ubiquitination process is inhibited, resulting in increased levels of ARTS. Increased ARTS in turn leads to a decrease of Bcl-2 and Bcl-xL protein levels, cytochrome c release from mitochondria and apoptosis.

Highlights

ARTS is a mitochondrial protein that promotes apoptosis induced by a variety of proapoptotic stimulators
ARTS is an unusual septin protein family member that is localized within mitochondria in living cells [10]
In response to proapoptotic stimuli, ARTS is released from mitochondria into the cytosol where it can bind to and inhibit IAPs such as XIAP [11]

Summary

MATERIALS AND METHODS

Chemicals and Reagents—Media, serum, antibiotics, and supplements were purchased from Biological Industries (Beit Haemek, Israel). Apoptotic Stimulation—For apoptosis assays, 40 h after the transient transfection cells were treated with different agents: TGF-␤ (10 ng/ml) for 24 h in medium containing 1% fetal calf serum, etoposide 50 ␮g/ml for 16 h, or staurosporine (1–1.2 ␮M) for 0 – 8 h. The cells were harvested by scraping the plate, washed twice with ice-cold 1ϫ PBS, and lysed using radioimmune precipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8), 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate containing protease inhibitors) (mini complete, Roche Applied Science). The cells were harvested, and 50 ␮g of protein lysate were separated on 12% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with anti-AU5 monoclonal antibodies. After centrifugation (16,000 ϫ g for 15 min at 4 °C), equal amounts of protein were taken for immunoprecipitation with monoclonal anti-ARTS antibodies (Sigma). The proteins were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, and visualized by exposing the membrane to film sensitive to 35S

Regulation of ARTS Levels through Ubiqutination

RESULTS

DISCUSSION