NUCLEAR-MEMBRANE VESICLE TARGETING TO CHROMATIN IN A DROSOPHILA EMBRYO CELL-FREE SYSTEM

A Drosophila cell-free system was used to characterize proteins that a re required for targeting vesicles to chromatin and for fusion of vesi cles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl ab olished their ability to bind to chromatin. Binding of salt-treated ve sicles to chromatin could be restored by adding the dialyzed salt extr act. Lamin Dm is one of the peripheral proteins whose activity was req uired, since supplying interphase lamin isoforms Dm(1) and Dm(2) to th e assembly extract restored binding. As opposed to the findings in Xen opus, okadaic acid had no affect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chrom atin even in the presence of the dialyzed salt extract. Once vesicles attached to chromatin surface, fusion events took place that were foun d to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin bu t not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GT Pase activity.