Figure 1.
Figure 1. C. elegansCYB-1 is a direct substrate of the CRL2ZYG-11 complex. (A) Egg hatching percentage ± SEM for zyg-11(ts) at the semipermissive temperature of 20°C with the listed RNAi treatments; from at least three independent counts, with the total number of embryos analyzed (from left to right): 667, 1,163, 448, and 222, respectively. (B) ZYG-11 negatively regulates CYB-1 levels in a proteasome-dependent manner. VSV-G–CYB-1 was coexpressed with FLAG–ZYG-11 or control FLAG–CYE-1 in HEK293T cells treated with human ZYG11A/B siRNA. Cells were treated for 8 h with nocodazole and (where indicated) the proteasome inhibitor MG132 for the last 4 h. Western blots of whole cell lysate were probed with the indicated antibodies. The relative VSV-G–CYB-1 to actin ratio is given below the top blot. Similar results were obtained in six independent experiments with varied cell treatments (± ZYG11A/B knockdown, ± nocodazole, and ± nocodazole and proTAME). (C) ZYG-11 and CYB-1 physically interact. VSV-G–CYB-1 was coexpressed in HEK293T cells with either FLAG–ZYG-11 or FLAG–CUL-4 in the presence of the proteasome inhibitor LLnL. Anti-FLAG antibody was used for IP, with analysis by Western blot (top two panels). Similar results were obtained in two independent experiments. (D) Endogenous ZYG-11 physically interacts with GFP::CYB-1 in C. elegans. Wild-type and animals expressing CYB-1::GFP were grown ± cul-2 RNAi. GFP::CYB-1 was immunoprecipitated and analyzed by Western blot (top two panels). (E) ZYG-11 physically interacts with the CBOX1 domain of CYB-1. FLAG–ZYG-11 was coexpressed with VSV-G–tagged CYB-1 truncations, NTER, CBOX1, or CBOX2 in HEK293T cells in the presence of MG132. FLAG–ZYG-11 was immunoprecipitated and analyzed by Western blot (top two panels). A diagram of the full-length and truncated CYB-1 proteins is shown at bottom. The asterisk marks the location of the residue homologous to the cyb-2.1 E120K zyg-11 mutant suppressor allele. (F) CBOX1 of CYB-1 is degraded during meiosis in the C. elegans embryo in a ZYG-11–dependent manner. Bar graph showing the levels ± SEM of GFP::CYB-1 truncations in two-cell stage embryos relative to the level in meiosis I–stage embryos (set to 100) in the RNAi treatments listed; from left to right, n = 5, 4, 12, 4, and 7, respectively. P-values are calculated for GFP levels in two-cell stage embryos versus the corresponding meiotic embryos. For all figures, asterisks above bars represent p-values relative to the control sample; asterisks above lines denote comparisons under the lines. ns, not significant; *, P < 0.05; **, P < 0.01.

C. elegansCYB-1 is a direct substrate of the CRL2ZYG-11 complex. (A) Egg hatching percentage ± SEM for zyg-11(ts) at the semipermissive temperature of 20°C with the listed RNAi treatments; from at least three independent counts, with the total number of embryos analyzed (from left to right): 667, 1,163, 448, and 222, respectively. (B) ZYG-11 negatively regulates CYB-1 levels in a proteasome-dependent manner. VSV-G–CYB-1 was coexpressed with FLAG–ZYG-11 or control FLAG–CYE-1 in HEK293T cells treated with human ZYG11A/B siRNA. Cells were treated for 8 h with nocodazole and (where indicated) the proteasome inhibitor MG132 for the last 4 h. Western blots of whole cell lysate were probed with the indicated antibodies. The relative VSV-G–CYB-1 to actin ratio is given below the top blot. Similar results were obtained in six independent experiments with varied cell treatments (± ZYG11A/B knockdown, ± nocodazole, and ± nocodazole and proTAME). (C) ZYG-11 and CYB-1 physically interact. VSV-G–CYB-1 was coexpressed in HEK293T cells with either FLAG–ZYG-11 or FLAG–CUL-4 in the presence of the proteasome inhibitor LLnL. Anti-FLAG antibody was used for IP, with analysis by Western blot (top two panels). Similar results were obtained in two independent experiments. (D) Endogenous ZYG-11 physically interacts with GFP::CYB-1 in C. elegans. Wild-type and animals expressing CYB-1::GFP were grown ± cul-2 RNAi. GFP::CYB-1 was immunoprecipitated and analyzed by Western blot (top two panels). (E) ZYG-11 physically interacts with the CBOX1 domain of CYB-1. FLAG–ZYG-11 was coexpressed with VSV-G–tagged CYB-1 truncations, NTER, CBOX1, or CBOX2 in HEK293T cells in the presence of MG132. FLAG–ZYG-11 was immunoprecipitated and analyzed by Western blot (top two panels). A diagram of the full-length and truncated CYB-1 proteins is shown at bottom. The asterisk marks the location of the residue homologous to the cyb-2.1 E120K zyg-11 mutant suppressor allele. (F) CBOX1 of CYB-1 is degraded during meiosis in the C. elegans embryo in a ZYG-11–dependent manner. Bar graph showing the levels ± SEM of GFP::CYB-1 truncations in two-cell stage embryos relative to the level in meiosis I–stage embryos (set to 100) in the RNAi treatments listed; from left to right, n = 5, 4, 12, 4, and 7, respectively. P-values are calculated for GFP levels in two-cell stage embryos versus the corresponding meiotic embryos. For all figures, asterisks above bars represent p-values relative to the control sample; asterisks above lines denote comparisons under the lines. ns, not significant; *, P < 0.05; **, P < 0.01.

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