Lysine residues in the Mult1 cytoplasmic tail inhibit protein expression. (A) Amino acid sequence of the Mult1 transmembrane (underlined) and cytoplasmic domains with lysines shown in bold. The boxes indicate duplicated amino acid sequences within the cytoplasmic tail, and arrows show the location of the C termini of the various deletion mutants. (B, C, and E) Fibroblasts were transduced using the same approach as in Fig. 1 C with WT Mult1, Mult1 truncation or KR mutants, or chimeras consisting of the H60a extracellular domain linked to the TM (transmembrane) and IC (intracellular) domains of Mult1 (TM+IC), or to the H60a TM domain and the Mult1 IC domain (IC tail only), and stained with the indicated antibodies. Histograms were gated on cells with similar GFP expression. (D) Fibroblasts were grown on coverslips, followed by permeabilization and staining with Mult1 antibody and PE conjugated secondary antibody. The complete absence of detectable Mult1 in cells transduced with WT Mult1 is likely caused by the lower sensitivity of immunofluorescence microscopy, as compared with flow cytometry (B). Bar, 10 µm. Data in this figure is representative of at least three independent experiments.

UV irradiation induces transduced Mult1 independently of the DNA damage response pathway. (A) Fibroblasts transduced with FLAG-tagged WT Mult1 were exposed to the indicated genotoxic stress treatments for 16 h, followed by staining with FLAG antibody and flow cytometry. Data are representative of three independent experiments. (B) Fibroblasts transduced with FLAG-tagged WT Mult1 were pretreated for 1 h with either 2 mM caffeine (top right) or 150 nM Chk1 inhibitor (SB218078; bottom right), followed by exposure to UV-C light and staining with FLAG antibodies 16 h later for analysis by flow cytometry. Solid gray histograms and dashed gray lines in A and B represent isotype staining of untreated and treated cells respectively. Data are representative of two independent experiments.

Heat shock induces Mult1 by altering protein half-life. (A) Fibroblasts transduced with FLAG-tagged Mult1 were shifted to 45°C for 30 min, followed by incubation at 37°C for 4 h. Cells were lifted with 5 mM EDTA and stained with FLAG antibody for flow cytometry. Cells treated in duplicate were used for Mult1 quantitative PCR and normalized to GAPDH. Data shown is mean ± range; n = 2. (B) Heat shock of untransduced fibroblasts and WEHI 7.1 cells was performed as in A, followed by 37°C incubation for the indicated time. Endogenous Mult1 levels were measured by flow cytometry. Cells treated in duplicate were used for Mult1 quantitative PCR as described in A. HS, heat shock. (C) B6 thymocytes were heat shocked as in A, followed by 37°C incubation for 6 h, staining with Mult1 antibody, and, in some cases, gating on large (forward scatter-high [FSC-high]) or small (FSC-low) cells. Identically treated cells were used for Mult1 quantitative PCR and normalized to 18S ribosomal RNA. Data shown is mean ± SD; n = 3. Solid gray histograms and dashed gray lines in A–C represent isotype staining of untreated and treated cells respectively. (D) Analysis of Mult1 protein half-life in heat shocked WEHI 7.1 cells. Cells were pulse labeled with radioactive amino acids and heat shocked at time zero of the chase as in A, followed by incubation at 37°C for the remainder of the chase. Staining of Mult1 on identically treated cells is shown at the 4-h time point. Cells from each time point were lysed in TNT lysis buffer, and Mult1 was immunoprecipitated and resolved by SDS-PAGE. Signal intensity was determined using ImageJ software (NIH). Data in this figure is representative of at least three independent experiments.

Mult1 protein is inefficiently expressed. (A) Quantitative RT-PCR for Mult1 was performed on cDNA prepared from the indicated tissues and cell lines. The relative amount of Mult1 RNA was determined by normalizing to GAPDH transcript levels. Data shown is mean ± SD; n = 3. (B) Thymocytes and YAC-1 cells were stained with Mult1 antibody and analyzed by flow cytometry. (C) B6 fibroblasts were transduced with a retroviral vector encoding the indicated FLAG-tagged protein and stained with FLAG antibody for analysis of gated GFP⁺ cells by flow cytometry. Data in this figure is representative of at least three independent experiments.

Ubiquitination is involved in the inhibition of Mult1 expression. (A) Fibroblasts transduced with FLAG-tagged Mult1 were lysed in RIPA buffer and Mult1 was immunoprecipitated with Mult1 or control antibody, followed by Western blotting with either ubiquitin or FLAG antibodies. Iso., isotype control; Vec., empty vector transduced; WT, WT Mult1 transduced; KR, Lys to Arg mutant Mult1 transduced. (B) Untransduced, WT Mult1–transduced, or KR Mult1–transduced fibroblasts were treated with either DMSO, 20 µM MG132, or 100 µM chloroquine for 6 h, followed by staining with FLAG antibody and analysis by flow cytometry. (C) Untransduced, WT Mult1–transduced, or KR Mult1–transduced fibroblasts were grown on tissue culture–treated glass slides and treated with DMSO, 20 µM MG132, or 100 µM chloroquine for 6 h. Cells were permeabilized, stained with Mult1 antibodies followed by Alexa Fluor 555–conjugated secondary antibodies and DAPI, and analyzed by immunofluorescence microscopy. Bar, 10 µm. Data in this figure is representative of at least four independent experiments.

Mult1 induction by UV irradiation is accompanied by a decrease in ubiquitination. (A) Fibroblasts transduced with FLAG-tagged Mult1 were exposed to 50 J/M² UV-C irradiation. After UV treatment for 4 or 8 h, cells were stained with FLAG antibody and analyzed by flow cytometry. Cells treated in duplicate were used for Mult1 quantitative PCR and normalized to GAPDH. Data shown is mean ± range; n = 2. (B) Untransduced fibroblasts were UV irradiated as in Fig. 1 A, followed by staining with Mult1 antibody at the indicated time after UV treatment. Cells treated in duplicate were used for Mult1 quantitative PCR as in A. (C) B6 fibroblasts transduced with H60a or the H60a-Mult1 chimera (the H60a extracellular domain with the TM and IC domains of Mult1) were UV irradiated as in Fig. 1 A, followed by staining with Mult1 or H60a antibody at 6 h after treatment. Parallel cultures of cells transduced with the H60a-Mult1 chimera were used for semiquantitative RT-PCR to quantify transcripts of the chimeric protein, endogenous Mult1, and HPRT. Solid gray histograms and dashed gray lines in A–C represent isotype staining of untreated and treated cells respectively. (D) Fibroblasts transduced with indicated retroviral construct were treated with 50 J/M² UV-C irradiation and scraped into RIPA buffer 6 h after UV treatment. Cleared lysates were immunoprecipitated with Mult1 antibody and Western blotted with FLAG or ubiquitin antibodies. Vec., Vector transduced; WT, WT Mult1 transduced; KR, Lys to Arg mutant Mult1 transduced cells. Data in this figure is representative of at least three independent experiments.