Importin-β and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function

0301 basic medicine importin beta GTPase-Activating Proteins SUMO-1 Protein Receptors, Cytoplasmic and Nuclear Exportin 1 Protein Karyopherins beta Karyopherins Nuclear Pore Complex Proteins 03 medical and health sciences CRM1/exportin 1 kinetochore function RANBP2 Humans CRM1/exportin 1; Importin-Î2; Kinetochore function; Proximity ligation assay; RANBP2; Cell Biology proximity ligation assay Kinetochores HeLa Cells Molecular Chaperones
DOI: 10.1242/jcs.197905 Publication Date: 2017-06-10T01:02:20Z
ABSTRACT
ABSTRACT Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2–SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2–SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.
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