Figure 1.
The chemically mutagenized clone, CHO-CDTRA2, is resistant to CDT and complemented by expression of Derl2.
Parental A745 cells, chemically induced mutant CHO-CDTRA2 cells, and CHO-CDTRA2 cells expressing Derl2 were seeded in a 384-well plate (1×103 cells/well) and allowed to adhere overnight, followed by 48 hour intoxication with Aa-CDT (a), Hd-CDT (b), Ec-CDT (c) and Cj-CDT (d) and quantitation of viability using ATPlite 1-step reagent (Perkin Elmer). Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.
Figure 2.
Derl2 is required for CDT intoxication.
Viability of parental A745TKR cells, retrovirally induced mutant CHO-CDTRC1 cells, and CHO-CDTRC1 cells expressing Derl2 after intoxication with Aa-CDT (a), Hd-CDT (b), Ec-CDT (c) and Cj-CDT (d). Intoxication was performed similar to Fig. 1. (e) Top: representation of the Derl2 open reading frame with boxes representing exons, gray arrows representing primers, and upside down triangles representing proviral insertions. Bottom: agarose gel of genomic PCR from parental A745TKR, CHO-CDTRC1 and CHO-CDTRF1 cells using primers detailed in the diagram. (f) Overexpression of Derl1 does not complement resistance to CDT. Derl2 deficient CHO-CDTRC1 cells expressing empty vector, Derl1, and Derl2 were intoxicated with Hd-CDT, similar to Fig. 1. (g) Derl2 was immunoprecipitated from normalized cell lysates and precipitated proteins analyzed by western blot with anti-Derl2 antibody. (h) CRISPR mediated deletion of Derl2 in HeLa cells causes resistance to Hd-CDT. HeLa cells were transfected with Cas9 DNA and gDNA, followed by selection with G418 and Hd-CDT. Following selection, wildtype and Derl2-deleted cells were intoxicated with Hd-CDT, similar to figure 1. (i) CRISPR mediated deletion of Derl2 results in decreased expression as judged by western blot of anti-Derl2 immunoprecipitated protein from normalized cell lysates. Increasing amounts of immunoprecipitated protein loaded for each condition, corresponding to input from 0.5, 1, or 2×106 cells. (j–l) Retrograde trafficking of Hd-CDT in Derl2 deficient cells is blocked at the endoplasmic reticulum. (j) A745TKR and CHO-CDTRC1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 10 or 60 minutes. Cells were then fixed and stained with DAPI (nuclei, blue), Concanavalin A (ER, red) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (k,l) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER marker. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.
Figure 3.
Hrd1 is required for CDT intoxication.
(a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.
Figure 4.
The interaction of Derl2 and p97 is not required for CDT intoxication.
(a) Derl2-GFP fails to bind p97, similar to Derl2ΔC. 293 cells were transfected with vectors encoding S-tagged versions of the indicated forms of Derl2. After 3 days, the cells were lysed and western blot was performed on S-protein precipitates with anti-p97 and anti-S-tag antibodies (b) Overexpression of Derl2-GFP does not affect Hd-CDT intoxication of parental A745TKR cells. Parental A745TKR cells expressing empty vector, Derl2 or Derl2-GFP were intoxicated with Hd-CDT, similar to Fig. 1. (c, d) Derl2-GFP and Derl2ΔC complement sensitivity to Hd-CDT in CHO-CDTRC1. CHO-CDTRC1 cells expressing empty vector, Derl2, (c) Derl2-GFP or (d) Derl2ΔC were intoxicated similar to Fig. 1. (e) Dominant negative p97 reduces sensitivity of 293 cells to Hd-CDT. 293 cells stably expressing TCRαGFP were transfected with plasmids encoding CD4 and either dominant negative (R586A) or control (R700A) p97, followed by intoxication with Hd-CDT for 48 hours and staining with Hoechst and anti-CD4 antibodies. Flow cytometry was performed to obtain geometric mean fluorescence values for TCRαGFP (GFP) in CD4+ cells and cell cycle profile of CD4 negative (grey shaded; untransfected control) and CD4 positive cells (black lines). (f) The Derl2 WR motif is not required for intoxication by Hd-CDT. CHO-CDTRC1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A or Derl2 T59A were intoxicated similar to figure 1. (g–i) Retrograde trafficking of Hd-CDT in p97 deficient cells is blocked at the endoplasmic reticulum. (g) Following transfection with pH2B-GFP (blue) and either dominant negative or control p97, wildtype and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with anti-Hd-CdtB (green) antibody and anti-calreticulin antibody (red). White scale bars indicate 5 µm. pH2B-GFP pseudo-colored blue; Hd-CdtB pseudo-colored green and calreticulin pseudo-colored red (h, i) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments, percent viability is normalized to unintoxicated controls and error bars indicate standard error.
Figure 5.
Identification of Derl2 domains required for CDT intoxication.
(a–c) CHO-CDTRC1 cells expressing empty vector (squares), Derl1-S (triangles), or Derl2-S (diamonds) were intoxicated in each panel, similar to Fig. 1, and compared to derlin variants indicated below. Anti-DERL1 (a) or anti-Derl2 (b, c) western blot of S-protein agarose precipitated protein from normalized cell lysates show expression levels of chimeric derlins. Cartoons depict Derl1 (black) and Derl2 (grey) sequences in each chimera. (a) CHO-CDTRC1 cells expressing Derl1-S (triangles, #1) or Derl21–187:Derl1189–251-S tag (circles, #2) were challenged with Hd-CDT. (b) CHO-CDTRC1 cells expressing Derl2-S (diamonds, #1), Derl21–112:Derl1114–121:Derl2120–239-S (circles, #2) or Derl21–161:Derl1163–171: Derl2171–239-S (inverted triangles, #3) were intoxicated as above. (c) CHO-CDTRC1 cells expressing Derl2-S (diamonds, #1), Derl11–88:Derl288–239-S (open boxes, 2), Derl11–138:Derl2138–239-S (open triangles, #3) or Derl11–162:Derl2162–239-S (open diamonds, #4) were intoxicated as above. Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.
Figure 6.
Derl2 and Hrd1 contribute to sensitivity to Ricin, independent of the Derl2 WR motif and the interaction of Derl2 with p97.
(a) Derl2 deficiency causes resistance to ricin. A745TKR cells, CHO-CDTRC1 cells, and CHO-CDTRC1 cells expressing Derl2 were seeded in a 384-well plate (1×103 cells/well) and allowed to adhere overnight, followed by 48 hour intoxication with ricin and quantitation of viability using ATPlite 1-step reagent (Perkin Elmer). Ricin LD50 values were calculated from three independent experiments and paired t-test was performed to calculate two tailed p-values. (b) CRISPR mediated Hrd1 deletion in 293 cells causes resistance to ricin. Wildtype and Hrd1-deleted 293 cells were intoxicated with ricin, similar to figure (a). (c) Derl2ΔC complements the resistance to ricin. CHO-CDTRC1 cells expressing empty vector, Derl2 and Derl2ΔC were intoxicated similar to (a). (d) The Derl2 WR motif is not required for intoxication by ricin. CHO-CDTRC1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A and Derl2 T59A were intoxicated similar to (a). Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.