The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo.
RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.
Joseph
Jomon
J
National Institutes of Health, National Institute of Child Health and Human Development, Building 18, Room 106, 18 Library Drive, MSC-5431, Bethesda, MD 20892, USA.
Liu
Song-Tao
ST
Jablonski
Sandra A
SA
Yen
Tim J
TJ
Dasso
Mary
M
D017931:DNA Primers; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D018386:Kinetochores / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D034622:RNA Interference; D025842:SUMO-1 Protein / Q000378:metabolism
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D007211:Indoles; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C092317:RANGAP1 protein, human; D025842:SUMO-1 Protein; C094493:ran-binding protein 2; C007293:DAPI