THE CELL CYCLE-DEPENDENT LOCALIZATION OF THE CP190 CENTROSOMAL PROTEIN IS DETERMINED BY THE COORDINATE ACTION OF 2 SEPARABLE DOMAINS

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, os cillates in a cell cycle-specific manner between the nucleus during in terphase, and the centrosome during mitosis. To characterize the regio ns of CP190 responsible for its dynamic behavior, we injected rhodamin e-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-laps e fluorescence confocal microscopy. A single bipartite 19-amino acid n uclear localization signal was detected that causes nuclear localizati on. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins conta ining distinct portions of this region show weaker centrosomal localiz ation. Fusion proteins that contain both nuclear and centrosomal local ization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only th e 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. Bo th 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-dependen t manner. Experiments in which colchicine is used to depolymerize micr otubules in the early Drosophila embryo demonstrate that both CP190 an d CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.