XIAP and MURR1 interactions in mammalian cells. (A) Coprecipitation of endogenous MURR1 and XIAP: 293 cells were lysed in a buffer containing 0.5% Triton X-100. Immunoprecipitation of endogenous proteins was performed using a rabbit polyclonal antibody against MURR1 or normal rabbit Ig. The precipitated material was used for a Western blot for XIAP (top panel) and the supernatants after immunoprecipitation were immunoblotted for MURR1 (middle panel) and β-actin (bottom panel). (B) Schematic of deletion constructs: truncations in XIAP as shown in this schematic were expressed in mammalian cells in fusion with GST and used as shown in (C). (C) Deletion mapping: 293 cells were cotransfected with C-terminal Flag-tagged MURR1 together with vectors expressing GST fusion proteins with XIAP full-length and various truncated forms as indicated. In addition, GST-3 × BIR wild-type, or GST-3 × BIR D148A/W310A were also used in these experiments (right panels). Cell lysates prepared with a buffer containing 0.5% Triton X-100 were used for precipitations with glutathione beads. The precipitated material was immunoblotted with Flag antibodies. (D) Coprecipitation of other 3-BIR containing IAPs: 293 cells were transfected with either MURR1–GST or GST together with Myc₆-c-IAP2 or Myc₆-NAIP. Cell lysates prepared with a buffer containing 0.5% Triton X-100 were used for precipitations using glutathione sepharose beads. The precipitated material was immunoblotted with Myc antibody or c-IAP1 antibody.

MURR1 does not affect the antiapoptotic activities of XIAP. Expression vectors encoding Bax (A and B) or Fas (C and D) were transfected into 293 cells either with empty vector controls or XIAP, MURR1 or both, together with a plasmid expressing EGFP as a marker of transfection. Cell viability was assessed by morphology of EGFP-positive cells 16 h (A) or 30 h (C) post-transfection, and caspase-3 activity was determined in the same cells (B and D). Data presented represent the average and s.e.m. of three measurements per group.

XIAP targets MURR1 for ubiquitination and degradation. (A) MURR1 protein levels are regulated by XIAP: 293 cells were transfected with pEBB–XIAP, pEBB–XIAP H467A or vector control or with siRNA oligonucleotides targeting GFP or XIAP. MURR1–Flag was cotransfected and expression was determined by Western blot using Flag antibodies. The same membrane was probed for β-actin and for XIAP. (B) MURR1 protein levels are regulated by proteasomal degradation: 293 cells were transfected with MURR1–Flag and protein levels were determined by Western blot using Flag antibodies. The cells were cotransfected with XIAP or a vector control and treated with lactacystin (10 μM) for the time indicated. (C) MURR1 is ubiquitinated in vitro and in intact cells: in vitro translated and ³⁵S-labeled MURR1 was used as substrate for in vitro ubiquitination (left). Ubiquitination was also studied in intact cells. 293 cells were transfected as indicated. One day after transfection, cells were treated with lactacystin (10 μM) for 8 h, and lysed in RIPA buffer. Lysates were precipitated with glutathione sepharose beads and analyzed by Western blot using ubiquitin antibodies (right). (D) Effect of XIAP on MURR1 ubiquitination: 293 cells were transfected as indicated. One day after transfection, cells were lysed in RIPA buffer and precipitation was performed using glutathione sepharose beads. The precipitated material was used for Western blot with ubiquitin antibodies. (E) Effect of XIAP in the accumulation of ubiquitinated MURR1 after lactacystin treatment: 293 cells were transfected as indicated; 1 day later, half of the cultures were treated with lactacystin (10 μM) for an additional 3 h. Cell lysates were prepared with RIPA buffer and precipitated with glutathione sepharose beads. The precipitated material was used for Western blot with ubiquitin antibodies. (F) Murr1 protein levels in Xiap-deficient fibroblasts: cell lysates from wild-type and Xiap-deficient fibroblasts were used for Western blot and immunoblotted as indicated.

Localization of fluorescently tagged MURR1. (A) Colocalization of fluorescently tagged MURR1 and XIAP: 293 cells plated onto coverglass chamber slides were transfected with MURR1 fused with YFP (MURR1–YFP) and XIAP fused with CFP (CFP–XIAP) (top panels), or MURR1–YFP and CFP (middle panels), or YFP and CFP–XIAP (lower panels). Counterstaining of the nuclei was also performed using Hoechst 33342 (displayed in red for clarity). The scale bar shown corresponds to 18 μm. (B) MURR1 does not colocalize with mitochodrial or lysosomal markers: MURR1–GFP was transfected into 293 cells seeded on coverglass chambered slides. After 24 h the cells were stained with the vital dyes Mitotracker (left panel) or Lysotracker (right panel) (Molecular Probes). Scale bars correspond to 18 μm.

Copper levels are regulated by XIAP. 293 cells were transiently transfected with siRNA oligonucleotides or plasmids as indicated. Copper levels were measured by atomic absorption and corrected for cell number in each sample. The effect of RNAi of MURR1 is shown in (A) and the effect of transient transfection of XIAP is shown in (B). Similar experiments were performed with transformed fibroblasts derived from wild-type and Xiap-deficient mice (C). Copper content in liver tissue obtained from Xiap-deficient mice and age- and sex-matched wild-type controls is presented in (D). Each graph depicts all data points from multiple independent experiments. The mean and s.e.m. bars are also shown. A Student's t-test was performed, and the P-value is indicated in the graph panel. Western blots for MURR1 or XIAP are included below each panel as indicated.

Non-K48 ubiquitination of MURR1. (A) Non-K48-linked ubiquitination of MURR1: 293 cells were transfected with either wild-type His₆-tagged ubiquitin or a K48R mutant of ubiquitin along with MURR1–Flag. In addition, XIAP wild-type, XIAP H467 or a vector control were also transfected, as indicated. Two days after transfection, cell lysates were prepared and used for precipitation with Ni–NTA agarose beads. The precipitated material was immunoblotted with Flag antibodies. Molecular weight markers are in kilodaltons. (B) Binding of XIAP H467A to MURR1 and the effects of proteasome inhibition: 293 cells were transfected as indicated and 1 day later half of the cultures were treated with lactacystin (10 μM) for an additional 6 h. Cell lysates were then prepared with RIPA buffer and precipitated with glutathione sepharose beads. The precipitated material was immunoblotted for XIAP. (C) Schematic representation of MURR1 ubiquitination pathways and the role of XIAP.