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

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 SUMOconjugated 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.

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays

“…PLA signals localized along the spindle MTs and at the interface between MT plus-ends and KTs in metaphase (Fig. 4c , top row); chromosomal regions were devoid of PLA signals, consistent with the notion that the KT-associated RANBP2 pool is free of importin beta-1 30 . In telophase, importin beta-1/RANBP2 PLA products relocalized around the decondensing chromatin, in the region where the NE and NPCs reassemble around the reforming nuclei (Fig.…”

“…Some actually act in mitosis, indicating “moonlighting” roles. We also depicted all components of the RRSU SUMO ligase complex, which is emerging as a temporal regulator of KT functions 28 , 30 , 31 , consistent with the established ability of importin beta-1 to interact with RANBP2 14 , 37 . Another interesting group comprises tubulin beta, the tubulin-binding protein NUSAP and importin-7, with which NUSAP also interacts.…”

“…In other cases, importin beta-1 displaces factors away from their site of action. For example, it prevents the unscheduled onset of KT functions by localizing certain regulatory factors (e.g., RANBP2) away from KTs 14 , 29 , 30 . Depicting the spatial map of importin beta-1 interactions with its partners can help to understand its modes of action in mitotic cells.…”

“…Importin beta-1 also localizes to the mitotic spindle MTs and poles 12 , and its interaction with RANBP2 prevents the RRSU premature localization at KTs prior to MT attachment 14 , 29 . Recent work has shown that this is essential to regulate the timing of SUMO conjugation of KT factors, and hence KT functions 30 , 31 . However, beyond individual studies of specific mitotic factors, a comprehensive view of importin beta-1 interactors in mitotic cells is currently lacking.…”

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

“…The mitotic functions of XPO1, like its nuclear export activity, seem to be the subject of careful regulation through mechanisms that include phosphorylation [61] and competition with importins [62] .…”