Fig 2: The acceleration of MCL1 degradation by ALG-2 relies on its interaction with Rpn3.a The expression level of Rpn3 in Rpn3-KO Jurkat cell line. The protein levels of Rpn3 and GAPDH are determined by western blotting. b The proliferation rate of Rpn3-KO Jurkat cells. The assay was started with 500,000 cells and measured by Countstar cell-counter system. Data shown were representative of three independent experiments. c The proteasome activity in Rpn3-KO cell line was impaired, but was rescued with re-expression of Rpn3. d, e ALG-2 loses the ability to accelerate MCL1 degradation in Rpn3-KO Jurkat cell line. Rpn3-KO Jurkat cells were infected with ALG-2 retrovirus (d) or sh-ALG-2 retrovirus (e) for 40 h, then treated with CHX and MG132 for another 8 h. Cells were collected and lysed in RIPA buffer for detection. f The effect of ALG-2 on the stability of MCL1 when Rpn3 was rescued in Rpn3-KO cell. Data shown were representative of three independent experiments. Error bars indicate SD. ns, no significance; ***P < 0.001.

Fig 3: ALG-2 accelerates MCL1 degradation.a The effect of ALG-2 on the ubiquitination of MCL1. HEK293T cells were transfected with GFP-MCL1, HA-Ubiquitin, with or without ALG-2 for 48 h and lysed by RIPA buffer. Cell lysates were immunoprecipitated with GFP-Trap sepharose beads and analyzed by HA antibody using western blot. b, c ALG-2 accelerates MCL1 degradation in Jurkat cells. The ALG-2-OX and ALG-2-KD Jurkat cells (b) or activated cells (c) were seeded in six-well plates and treated with CHX, CQ or MG132 for 8 h, and lysed in RIPA buffer for detection.

Fig 4: ALG-2 is a potential regulator of proteasome activity.a The sketch map to illustrate the assay of GST pull-down coupled with LC-MS/MS. Jurkat cells were infected with GST or GST-Rpn3 retrovirus for 72 h and lysed by NP-40 buffer. The samples were prepared by GST pull-down and analyzed by LC-MS/MS. b The interaction of Rpn3 with candidate protein ALG-2 was validated by His-tag pull-down in prokaryotic expression system. Purified proteins were detected by SDS-PAGE with coomassie blue staining. c ALG-2 interacts with Rpn3 in vivo. HEK293T cells were co-transfected with GFP-Rpn3 and HA-ALG-2 for 48 h and lysated by RIPA buffer. Cell lysates were immunoprecipitated with GFP-Trap sepharose beads and analyzed by western blot. d ALG-2 interacts with Rpn3 in endogenous expression levels. HEK293T cells were transfected with HA-Rpn3 or HA-ALG-2 for 48 h and lysated by RIPA buffer. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. e ALG-2 and Rpn3 is dependent on the presence of Ca2+. f, g Colocalization of ALG-2 and Rpn3. HeLa cells were transfected with GFP-Rpn3 and RFP-ALG-2 (f) or GFP-Rpn3 only. Endogenous ALG-2 was analyzed by immunostaining with TRITC (g). Fluorescence was imaged by confocal laser scanning microscope. Scale bars: 10 μm.

Fig 5: ALG-2 enhances the activity of the proteasome.a, b The expression levels of ALG-2 in ALG-2-OX and ALG-2-KD Jurkat cell lines. The protein levels of ALG-2 and GAPDH are determined by western blotting. c, d ALG-2 enhances the activity of proteasome in Jurkat cells. The ALG-2-OX and ALG-2-KD Jurkat cells, either inactivated or activated, were incubated in digitonin permeabilization buffer with slight sonication. Cell lysates were incubated for 1 h at 37 °C in reaction buffer with Suc-LLVY-AMC. The activity of proteasome was quantitated by AMC fluorescence intensity in a microtiter plate reader at λEX = 380 nm/λEM = 460 nm. e ALG-2 enhances the activity of proteasome in stimulated PBMCs. PBMCs were infected with ALG-2 retrovirus (left panel) or sh-ALG-2 retrovirus (right panel) for 40 h and activated by PMA and ionomycin for 10 h. Then the cells were measured the activity of proteasome as c. f ALG-2 enhances the activity of proteasome dependent on its calcium-binding sites. ALG-2-KD Jurkat cells were infected with ALG-2 WT, ALG-2 E47D, ALG-2 E114D, and ALG-2 D169A retrovirus for 48 h, then activated by PMA and ionomycin for 10 h. Proteasomal activities were assessed by Suc-LLVY-AMC cleavage as c. g Co-immunoprecipitation assays of ALG-2 mutants with Rpn3. Data shown were representative of three independent experiments. Error bars indicate SD. **P < 0.01; ***P < 0.001.