ASSEMBLY OF THE NUCLEAR-PORE - BIOCHEMICALLY DISTINCT STEPS REVEALED WITH NEM, GTP-GAMMA-S, AND BAPTA

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xeno pus eggs to examine the process of nuclear pore assembly in vitro. Wit h this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nu clear pore requires the prior assembly of a double nuclear membrane. I nhibition of nuclear vesicle fusion by pretreatment of the membrane ve sicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block po re assembly. This indicates that NEM inhibits a single step in pore as sembly-the initial fusion of vesicles required to form a double nuclea r membrane. The presence of GTP gamma S blocks pore assembly at two di stinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fus ed double nuclear membranes. Interestingly, when the Ca2+ chelator BAP TA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nucl ear membrane, but devoid of nuclear pores. To order the positions at w hich GTP gamma S and BAPTA interfere with pore assembly, a novel ancho red nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gam ma S-sensitive step. Thus, through use of an in vitro nuclear reconsti tution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.