Degradation of FAT10 requires ubiquitination. (A) HEK-293 cells were transfected with pCS2-HA-FAT10-GV along with pCAGGS-FLAG-ubiquitin (FLAG-Ub) or pCAGGS-FLAG-lysine-less ubiquitin (FLAG-UbK0). After 48 h, cells were treated with CHX, and MG132 was added as indicated. Cells were harvested at the indicated times, and whole-cell lysates were subjected to SDS–PAGE and WB with the indicated antibodies as described in Materials and Methods. (B) CHO E36 and ts20 cells were transfected with pCS2-HA-FAT10-GV. After 42 h they were further incubated for 30 min at the permissive (30°C) or restrictive (43°C) temperature as described in Materials in Methods. CHX was added and the incubation continued at 30°C in the presence or absence of MG132. Cells were harvested at the indicated times, and whole-cell lysates were subjected to SDS–PAGE and WB with the indicated antibodies as described in Materials and Methods.

FAT10 can serve as a degradation signal. (A) HEK-293 cells were transfected with the indicated constructs and treated for the indicated times with CHX. MG132 was added as indicated along with CHX. Whole-cell lysates were resolved via SDS–PAGE and proteins visualized after WB with anti-actin and anti-GFP (i) or anti-HA (ii) antibodies. Numbers in (ii) denote the percentage of HA-FAT10-GV-GFP remaining compared with time 0. (B) HEK-293 cells were cotransfected with pCS2-GFP and pCS2-FAT10-GV-GFP. After 44 h MG132 was added for the indicated times, following which cells were lysed. Cell lysates were resolved via SDS–PAGE and proteins visualized after WB with an anti-GFP antibody. Numbers denote the level of FAT10-GV-GFP in the lysates compared with time 0. (C) HEK-293 cells were cotransfected with pCAGGS-FLAG-Ub along with pCS2-FAT10-GV-GFP or pCS2-FAT10-K0-GV-GFP. After 44 h, MG132 was added for 4 h, and the cells were lysed in RIPA buffer. Cell lysates were resolved via SDS–PAGE and proteins visualized after WB with the indicated antibodies. Molecular weight markers are indicated. (D) HEK-293 cells were transfected with pCS2-HA-GFP or pCS2-HA-FAT10-GV-GFP. After 48 h, the cells were lysed and the lysates subjected to the immunoprecipitation as described in the legend to Figure 1B. Total and immunoprecipitated proteins were resolved by SDS–PAGE and visualized after WB using the indicated antibodies. Molecular weight markers are indicated. (E) HEK-293 cells were cotransfected with pCS2-GFP and pCS2-FAT10-GV-GFP along with pCAGGS-FLAG-Ub or pCAGGS-FLAG-UbK0. After 44 h, CHX was added for the indicated times, after which cells were lysed. MG132 was added along with CHX when indicated. Cell lysates were resolved via SDS–PAGE and proteins visualized after WB with the indicated antibodies. Numbers indicate the percentage of FAT10-GV-GFP remaining compared with time 0.

Proteasomal degradation of FAT10. (A) HEK-293 cells were treated for 24 h with TNFα and IFNγ to induce endogenous FAT10. (i) When indicated, cells were treated with cycloheximide (CHX) and MG132. (ii) After induction, cells were treated with CHX in the presence or absence of MG132 for the indicated times. Whole-cell lysates were resolved electrophoretically and subjected to WB with the indicated antibodies as described in Materials and Methods. (B) HEK-293 cells were transfected with pCS2-HA-DHFR or various pCS2-HA-FAT10 constructs as indicated. After 48 h, cells were lysed, and whole lysates were subjected to immunoprecipitation, using immobilized anti-HA. Total and immunoprecipitated proteins were analyzed by WB after SDS–PAGE using the indicated antibodies. Molecular weight markers and immunoglobulin light chains (*) are indicated. (C) HEK-293 cells were transfected with pCS2-HA-FAT10-GV or pCS2-HA-FAT10-K0-GV. (i) CHX was added to inhibit protein synthesis, and MG132 was added as indicated to inhibit the proteasome. (ii) A similar experiment aimed at monitoring the effect of proteasome inhibition without inhibiting protein synthesis. Proteins were detected as described for A. Numbers in (i) indicate the percentage of HA-FAT10 remaining at the different time points compared with time 0. (D) HEK-293 cells were transfected as in B, and newly synthesized proteins were radiolabeled as described in Materials and Methods. The labeling was carried out at 30 or 37°C for 15 or 45 min, and MG132 was added as indicated. Cells were lysed in RIPA buffer, and RIPA-insoluble material was dissolved by boiling in sample buffer. (i) HA-tagged FAT10 molecules were immunoprecipitated and analyzed by SDS–PAGE and autoradiography. The proportion of FAT10-associated radioactivity in the soluble and insoluble fractions is presented quantitatively in C, i. (ii) Representation of the time- and temperature-dependent accumulation of WT and lysine-less (K0) HA-FAT10-GV in the insoluble fraction. (E) HEK-293 cells were transfected as in B and treated at 30 or 37°C during 4 h with CHX. MG132 was added as indicated. Whole-cell lysates were resolved via SDS–PAGE and subjected to WB with the indicated antibodies as described in Materials and Methods. Numbers denote the percentage of remaining HA-FAT10 compared with time 0.

FAT10 is ubiquitinated both in a cell-free system and in cells. (A) In vitro translated HA-FAT10-GV, HA-FAT10(1-86) (N-terminal half of FAT10, referred to as N), and HA-FAT10(87-165)-GV (C-terminal half of FAT10, referred to as C) were subjected to a cell-free ubiquitination assay as described in Materials and Methods. Ubiquitination was carried out in JY or HeLa cell extracts as indicated. The ubiquitination reactions were analyzed by autoradiography following SDS–PAGE. Molecular weight markers are indicated. (B) Ubiquitination of in vitro translated, ³⁵S-methionine-labeled HA-FAT10-GV was carried out in HeLa cell extract in the absence (−) or presence of WT (+), methylated (Me), or lysine-less (K0) ubiquitin. Proteins were resolved and visualized as described for A. Bands corresponding to FAT10 conjugated with one to five ubiquitin molecules are indicated on the left, and molecular weight markers are indicated on the right. (C) (i) HEK-293 cells were cotransfected with pCAGGS-FLAG-Ub along with pCS2-HA-FAT10-GV or pCS2-HA-FAT10-K0-GV. After 45 h, cells were treated for 3 h with MG132 (MG) as indicated and lysed. Proteins were immunoprecipitated with an antibody directed against FLAG, resolved by SDS–PAGE, and detected following WB with an anti-HA antibody as described in Materials and Methods. (ii) Same as (i), except that cells were transfected also with pCS2-HA-DHFR as a control, and proteins were first immunoprecipitated with anti-HA antibody and then detected by anti-HA (top) or anti-FLAG (bottom) antibody. Ubiquitinated FAT10 molecules (Ub-FAT10), immunoglobulin light (*) and heavy (**) chains, and molecular weight markers are indicated.

Apoptosis induces ubiquitination and degradation of FAT10. (A) Endogenous FAT10 expression was induced in HEK-293 cells by TNFα and IFNγ as described in Materials and Methods. STS was added along with MG132 (when indicated), and the cells were further incubated for the indicated times. Cells extracts were resolved via SDS–PAGE and proteins visualized following WB using the indicated antibodies. Positions of full-length (FL) and cleaved PARP-1 and the 100-kDa molecular weight marker are indicated. (B, C) HEK-293 cells were transfected with pCS2-HA-FAT10-GV and were treated for the indicated times with CHX or STS (B) or CHX with and without STS (C). The proteasome inhibitors MG132 (B and C, i) and epoxomicin (C, ii) were added as indicated. Cell lysates were resolved via SDS–PAGE, and proteins visualized after WB using the indicated antibodies. (D) HEK-293 cells were cotransfected with pCAGGS-FLAG-Ub and pCS2-HA-FAT10-GV or pCS2-HA-FAT10-K0-GV and were treated for 3 h with MG132 in the presence or absence of STS. RIPA lysates of the cells were then subjected to immunoprecipitation using immobilized anti-HA. Total and immunoprecipitated proteins were resolved via SDS–PAGE and proteins visualized after WB using the indicated antibodies. Molecular weight markers and immunoglobulin light chains (*) are indicated. (E) HEK-293 cells were transfected with pCS2-HA-FAT10-GV or pCS2-HA-FAT10-K0-GV and were treated for the indicated times with STS in the presence or absence of MG132. Cell lysates were resolved via SDS–PAGE and proteins visualized after WB using the indicated antibodies. (F) HEK-293 cells transfected with pCS2-FAT10-GV-GFP were treated and analyzed as in E. Numbers in B, C, E, and F indicate the percentage of remaining FAT10 (B, C, E) or FAT10-GFP (F) compared with time 0.