Figure 5.
NEDD1 specifically binds to MZT1/GCP3 NTE subcomplex of γ-TuRC. (A and B) Streptavidin pull-down assays with extracts of nocodazole-arrested mitotic HEK293T cells expressing Bio-NEDD1 (bait) and γ-TuRCΔNEDD1-GFP (prey), together with (+) or without (−) untagged CDK1/cyclin B (A) or HA-PLK1 (B), treated with (+) or without (−) RO3306 (A) or BI2536 (B), analyzed by Western blotting with indicated antibodies. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with GCP2/GCP3-GFP-Bio/γ-tubulin, GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin, GCP2/GCP3-GFP-Bio/γ-tubulin/MZT1, or GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin/MZT1 (bait), analyzed by Western blotting with indicated antibodies. (D) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with GCP2-GFP-Bio/γ-tubulin, GCP2-GFP-Bio/γ-tubulin/MZT2, GCP3-GFP-Bio/γ-tubulin, or GCP3-GFP-Bio/γ-tubulin/MZT1 (bait), analyzed by Western blotting with indicated antibodies. (E) Schematic overview of the domain organization of GCP3 and the deletion mutants, and summary of their interactions with NEDD1. The green ellipse represents MZT1. NTE, N-terminal extension. (F) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) and Bio-GFP–tagged GCP3-NTEs together with or without MZT1-GFP (bait), analyzed by Western blotting with indicated antibodies. The asterisks indicate the degradation bands of GCP3-NTE3. (G) Schematic overview of the domain organization of NEDD1 and the deletion mutants, and summary of their interactions with GCP3/MZT1. WD40, WD-40 repeat domain. (H) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing GFP-tagged NEDD1 full length (FL) or indicated deletion mutants (prey) and GCP3-NTE3-GFP-Bio together with or without untagged MZT1 (bait), analyzed by Western blotting with GFP antibody. (I) Model of the interaction between NEDD1 and GCP3/MZT1 in the context of the γ-TuRC complex. The dashed line surrounding half of the cone-shaped γ-TuRC, where GCP3 was positioned, indicates the asymmetric arrangement of NEDD1 within γ-TuRC.

NEDD1 specifically binds to MZT1/GCP3 NTE subcomplex of γ-TuRC. (A and B) Streptavidin pull-down assays with extracts of nocodazole-arrested mitotic HEK293T cells expressing Bio-NEDD1 (bait) and γ-TuRCΔNEDD1-GFP (prey), together with (+) or without (−) untagged CDK1/cyclin B (A) or HA-PLK1 (B), treated with (+) or without (−) RO3306 (A) or BI2536 (B), analyzed by Western blotting with indicated antibodies. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with GCP2/GCP3-GFP-Bio/γ-tubulin, GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin, GCP2/GCP3-GFP-Bio/γ-tubulin/MZT1, or GCP4/GCP5/GCP6-GFP-Bio/γ-tubulin/MZT1 (bait), analyzed by Western blotting with indicated antibodies. (D) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with GCP2-GFP-Bio/γ-tubulin, GCP2-GFP-Bio/γ-tubulin/MZT2, GCP3-GFP-Bio/γ-tubulin, or GCP3-GFP-Bio/γ-tubulin/MZT1 (bait), analyzed by Western blotting with indicated antibodies. (E) Schematic overview of the domain organization of GCP3 and the deletion mutants, and summary of their interactions with NEDD1. The green ellipse represents MZT1. NTE, N-terminal extension. (F) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) and Bio-GFP–tagged GCP3-NTEs together with or without MZT1-GFP (bait), analyzed by Western blotting with indicated antibodies. The asterisks indicate the degradation bands of GCP3-NTE3. (G) Schematic overview of the domain organization of NEDD1 and the deletion mutants, and summary of their interactions with GCP3/MZT1. WD40, WD-40 repeat domain. (H) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing GFP-tagged NEDD1 full length (FL) or indicated deletion mutants (prey) and GCP3-NTE3-GFP-Bio together with or without untagged MZT1 (bait), analyzed by Western blotting with GFP antibody. (I) Model of the interaction between NEDD1 and GCP3/MZT1 in the context of the γ-TuRC complex. The dashed line surrounding half of the cone-shaped γ-TuRC, where GCP3 was positioned, indicates the asymmetric arrangement of NEDD1 within γ-TuRC.

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