Assembly of the Nuclear Pore : Biochemically Distinct Steps Revealed with NEM , GTP / 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 Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP~S, and the Ca ÷+ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assemb l y t h e initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP~/S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca 2÷ chelator B A P T A 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 nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP~/S and B A P T A interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP~/S-sensitive step. Thus, through use of an ~ vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly. N 'UCLEAR pore complexes are critical to many aspects of nuclear function, yet the molecular structure of the pore and the mechanism of its assembly are poorly understood. The physical structure of the pore, estimated to be 125 million Da in mass, involves a permanent and specific membrane hole connecting the two nuclear membranes, with ~1,000 protein molecules (~60-100 different proteins) assembled within this hole to create the pore complex (Reichelt et al., 1990; Pante and Aebi, 1994; for reviews see Forbes, 1992; Gerace, 1992; Osborne and Silver, 1993; Fabre and Hurt, 1994; Rout and Wente, 1994). The great size of the pore clearly contributes to the complex nature of the assembly process, but an additional complexity is the need to establish structural and functional polarity to the pore, such that nuclear proteins are imported and mRNAs are exported. The structure of the nuclear pore becomes disassembled at mitosis. Immunofluorescence studies on cultured cells with anti-pore antibodies indicate that both the peripheral and integral membrane proteins of the pore distribute Please address all correspondence to Dr. D. J. Forbes, Department of Biology 0347, University of California San Diego, La Jolla, CA 92093-0347. Tel. : (619) 534-3398. Fax: (619) 534-0555. throughout the cell during mitosis (Gerace et al., 1982; Davis and Blobel, 1986; Park et al., 1987; Chaudhary and Courvalin, 1993; Cordes et al., 1993; Radu et al., 1993; Byrd et al., 1994). Reassembly of the nuclear envelope and nuclear pore is presumed to occur in a hierarchical fashion, with the assembly of the first components creating the target for binding and subsequent assembly of the later components, until a complete nuclear envelope is formed. For the nuclear pore, with only a few exceptions (Chaudhary and Courvalin, 1993; Byrd et al., 1994), the order and manner of this assembly is largely unknown. Cell-free nuclear reconstitution systems have proven quite useful for identifying distinct biochemical steps in both the mitotic breakdown and reassembly of nuclei. When chromatin from demembranated Xenopus sperm is mixed with the soluble and vesicular components of egg lysates, nuclei spontaneously assemble (Lohka and Masui, 1983). The resulting nuclei import nuclear-targeted proteins, replicate their DNA, and break down in response to mitotic signals (Lohka and Masui, 1984; Lohka and Mailer, 1985; Blow and Laskey, 1986; Finlay et al., 1987; Newport, 1987; see Newmeyer and Wilson, 1991, and references therein). When condensed sperm chromatin is added to an assembly reaction, the first step in nuclear assembly is a swelling or decondensation of the chromatin. This decon© The Rockefeller University Press, 0021-9525/96/01/5/16 $2.00 The Journal of Cell Biology, Volume 132, Numbers 1 & 2, January 1996 5-20 5 on A ril 3, 2017 D ow nladed fom Published January 1, 1996