title: Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis.
abstract: Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.
authors.forename: Nina
authors.initials: N
authors.lastname: Schweizer
authors.forename: Cristina
authors.initials: C
authors.lastname: Ferrás
authors.forename: David M
authors.initials: DM
authors.lastname: Kern
authors.forename: Elsa
authors.initials: E
authors.lastname: Logarinho
authors.forename: Iain M
authors.initials: IM
authors.lastname: Cheeseman
authors.forename: Helder
authors.initials: H
authors.lastname: Maiato
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins / Q000378:metabolism / Q000502:physiology*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C073597:TPR protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C075840:TTK protein, human