Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration.
The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107-160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin "global positioning system"or "GPS" pathways that are mechanistically parallel.
Section of Cell and Developmental Biology, Division of Biological Sciences 0347, University of California-San Diego, La Jolla, CA 92093-0347 Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041.
Cyril
C
Bernis
Beth
B
Swift-Taylor
Matthew
M
Nord
Sarah
S
Carmona
Yuh Min
YM
Chook
Douglass J
DJ
Forbes
D000595:Amino Acid Sequence; D000818:Animals; D002457:Cell Extracts; D002843:Chromatin / Q000378:metabolism; D048749:Cytokinesis; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D028884:Karyopherins / Q000737:chemistry / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D008941:Spindle Apparatus / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D002457:Cell Extracts; D002843:Chromatin; D029721:Drosophila Proteins; D028884:Karyopherins; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D010455:Peptides; C000592699:Tnpo protein, Drosophila; D029867:Xenopus Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein