The nuclear export factor Xpo1p targets Mad1p to kinetochores in yeast

Abstract

Nuclear pore complexes (NPCs) mediate all nucleocytoplasmic traffic and provide docking sites for the spindle assembly checkpoint (SAC) protein Mad1p. Upon SAC activation, Mad1p is recruited onto kinetochores and rapidly cycles between NPCs and kinetochores. We examined the mechanism of Mad1p movement onto kinetochores and show that it is controlled by two components of the nuclear transport machinery, the exportin Xpo1p and Ran–guanosine triphosphate (GTP). Mad1p contains a nuclear export signal (NES) that is recognized by Xpo1p. The NES, Xpo1p, and RanGTP are all required for Mad1p recruitment onto kinetochores in checkpoint-activated cells. Consistent with this function, Xpo1p also accumulates on kinetochores after SAC activation. We have also shown that Xpo1p and RanGTP are required for the dynamic cycling of Mad1p between NPCs and kinetochores in checkpoint-arrested cells. These results reveal an important function for Xpo1p in mediating intranuclear transport events and identify a signaling pathway between kinetochores and NPCs.

Nuclear transport and the mitotic apparatus: an evolving relationship

“…Thus, the localization of Crm1 at kinetochores appears independent of Ran-GTP or NES-containing proteins. This situation in metazoans is in contrast to that observed during closed mitosis in S. cerevisiae where, as mentioned in the previous section, SACcheckpoint-induced association of Xpo1 with kinetochores is dependent on the NES-containing Mad1 [31]. Recent studies in metazoan cells instead suggest that Crm1 is anchored to the kinetochore, either directly or indirectly through an interaction with another group of NPC components, the Nup107-160 complex ([53]; Fig.…”

“…Strikingly, in S. cerevisiae, most of the Mad1 ([60%) within these two pools is dynamic and exchanges rapidly between the two locations. This cycling process is controlled by the nuclear export factor, Xpo1 (the S. cerevisiae homologue of vertebrate Crm1), and RanGTP [31]. Xpo1 binds to Mad1 and both facilitates its targeting to kinetochores and its exchange between these structures and the Mlp sites at the NPC.…”

Phosphorylation of Crm1 by CDK1-cyclin B promotes Ran-dependent mitotic spindle assembly

“…Furthermore, in yeast, the spindle assembly checkpoint (SAC) protein Mad1p is recruited onto the kinetochore upon SAC activation by Xpo1p/Crm1 and RanGTP. Mad1p contains a functional nuclear export signal (NES) that bind to Xpo1p, forming a trimeric complex with RanGTP (Scott et al, 2009). Since Crm1 is highly conserved among species from yeast to humans, it may be evolutionarily common that Crm1 contributes to spindle assembly by recruiting NES-containing SAFs to the spindle or kinetochore.…”

Dual personality of Mad1

“…Furthermore, critical components of SAC signaling, specifically Mad1p and Mad2p, reside at NPCs in cycling cells, and, in response to KT-MT detachment, both Mad1p and Mad2p translocate from NPCs to KTs during the initial stage of SAC activation. [8][9][10] While Mad2p exhibits wholesale translocation to KTs, Mad1p localizes to both NPCs and KTs 8,11,12 and dynamically cycles between these molecular platforms during SACmediated arrest. 11,12 These observations raised the possibility that the KTIP and SAC were linked through the Mad1p-Mad2p complex.…”

“…11 Also contributing to the KT targeting of Mad1p is the nuclear transport factor Xpo1p. 12 Xpo1p normally functions to export proteins from the nucleus, 20 however, it has also been shown to play a role in directing Mad1p to KTs following SAC activation. 12 This Xpo1p function appears to be dispensable for SAC activation 12 but essential for the KTIP.…”