The importin-beta P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope.
Three of the four independently induced Ketel(D) dominantnegative female sterile mutations that identify the Drosophila importin-beta gene, originated from a C4114--> T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro-->Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-beta protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-beta does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-beta.
Timinszky
Gyula
G
The University of Szeged, Faculty of Medicine, Department of Biology, Somogyi B. u. 4, H-6720 Szeged, Hungary.
Tirián
László
L
Nagy
Ferenc T
FT
Tóth
Gábor
G
Perczel
András
A
Kiss-László
Zsuzsanna
Z
Boros
Imre
I
Clarke
Paul R
PR
Szabad
János
J
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D002942:Circular Dichroism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000033:anatomy & histology / Q000196:embryology*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007247:Infertility, Female / Q000235:genetics; D034882:Lamins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D008941:Spindle Apparatus / Q000378:metabolism; D019076:Transgenes; D028961:beta Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D002843:Chromatin; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; C424162:Fs(2)Ket protein, Drosophila; D034882:Lamins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein