The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains.
CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
K
K
Oegema
W G
WG
Whitfield
B
B
Alberts
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D008869:Microtubule-Associated Proteins; D009838:Oligodeoxyribonucleotides