Abstract
ABSTRACTIn metazoa, the Nup107 complex (also known as the nucleoporin Y-complex) plays a major role in formation of the nuclear pore complex in interphase and is localised to kinetochores in mitosis. The Nup107 complex shares a single highly conserved subunit, Seh1 (also known as SEH1L in mammals) with the GATOR2 complex, an essential activator of mTORC1 kinase. mTORC1/GATOR2 has a central role in the coordination of cell growth and proliferation. Here, we use chemical genetics and quantitative chromosome proteomics to study the role of the Seh1 protein in mitosis. Surprisingly, Seh1 is not required for the association of the Nup107 complex with mitotic chromosomes, but it is essential for the association of both the GATOR2 complex and nucleoporin Nup153 with mitotic chromosomes. Our analysis also reveals a role for Seh1 at human centromeres, where it is required for efficient localisation of the chromosomal passenger complex (CPC). Furthermore, this analysis detects a functional interaction between the Nup107 complex and the small kinetochore protein SKAP (also known as KNSTRN).
Highlights
The nuclear pore complex (NPC) is a large macromolecular assembly anchored at the nuclear envelope that allows transport between the nucleus and the cytoplasm during interphase (Hurt and Beck, 2015)
CRISPR-Cas9 gene editing technology allows efficient knock-in of specific sequences at a targeted locus (Cong et al, 2013; Mali et al, 2013). We employed both of these technologies (Natsume et al, 2016) to establish an HCT116 cell line in which endogenous Seh1 protein is tagged at the C-terminus with mini auxininducible degron (AID)-monomeric Clover (Fig. 1A,B)
As expression of Seh1– mini AID-monomeric Clover (mAIDmC) was under the control of the endogenous promoter for the Seh1 gene, the levels of Seh1–mAIDmC were similar to the Seh1 levels in the parental HCT116 cell line (Fig. 1E)
Summary
The nuclear pore complex (NPC) is a large macromolecular assembly anchored at the nuclear envelope that allows transport between the nucleus and the cytoplasm during interphase (Hurt and Beck, 2015). An initial observation suggesting that there could be an alternative function for the nucleoporins was finding that a subset of NPC components localised to the kinetochores of mitotic chromosomes (Belgareh et al, 2001; Hetzer and Wente, 2009; Joseph et al, 2002; Loiodice et al, 2004; Strambio-De-Castillia et al, 2010; Zuccolo et al, 2007). The nucleoporins that localise to kinetochores are members of the evolutionarily conserved
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