Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

Abstract

Abstract. Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP~/S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGAsepharose--depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein-containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae. T HE nuclear pore is responsible for establishing the distinct nuclear and cytoplasmic compartments of the eukaryotic cell. The pore is a highly selective channel through which macromolecular traffic must pass to enter or exit the nucleus. Although ions and other small molecules freely diffuse through the pore, larger molecules require a specific targeting signal for transit (for reviews, see Goldfarb and Michaud,.1991;Forbes, 1992;Gerace, 1992;Osborne and Silver, 1993;Powers and Forbes, 1994).The nuclear pore complex is a large and elaborate structure of ~120 million daltons and is comprised of N1,000 proteins. Structurally, it appears to consist of three ringsThe first two authors, Eva Meier and Brian R. Miller, contributed equally to the results presented here.Please address all correspondence to Douglass J. Forbes, Department of Biology, University of California at San Diego, La,lolla, CA 92093-0347. Tel.: (619) 534-0555....

Pre-M Phase-promoting Factor Associates with Annulate Lamellae inXenopusOocytes and Egg Extracts

“…AL membranes are continuous with and embedded within the ER (this study, Figure 6, A-C;Kessel, 1992;Terasaki et al, 2001), and it is not clear why they separate from the rest of the ER during fractionation, although the high concentration of nuclear pores is likely to significantly affect their density. AL accumulate in interphase Xenopus egg extracts (Dabauvalle et al, 1991;Meier et al, 1995) and disassemble in parallel with the nuclear envelope at mitosis/meiosis (Cordes et al, 1996;Imreh and Hallberg, 2000;Terasaki et al, 2001) and thus will be maximally accumulated at the time we made our extracts, favoring their identification in our study. We were able to confirm that part of the MPF population in the undisturbed cell colocalizes with AL by two independent visualization methods applied to whole oocytes: immunofluorescence on fixed material and live imaging of GFP-CyclinB2.…”

“…The resultant high-speed supernatant (HSS) was removed by side puncture of the tube with a syringe and wide gauge needle. At least three distinct layers of high-speed pellet were distinguishable: a dense orange pellet, presumed to be glycogen (Meier et al, 1995) overlain successively by a dark brown pellet (HSP-1a) and a yellow layer (HSP-1b). HSP-1a and -1b were removed with wide-mouthed pipette tips and swirled into EB containing protease inhibitors in 5-ml centrifuge tubes.…”

“…Micrographs were taken with a Hitachi H-600 electron microscope. Negative staining was carried out according to the protocol of Meier et al (1995) with modifications. A 10-l sample of unfixed HSP-2 was washed in 50 l of ice-cold wash buffer (10 mM HEPES pH 7.4, 3 mM MgCl 2 containing protease inhibitors as for EB; see above) in a 1.5-ml Eppendorf tube and centrifuged in a benchtop centrifuge at 4°C for 5 min at 14,000 rpm.…”

Association of the Human SUMO-1 Protease SENP2 with the Nuclear Pore

“…GST⅐SENP2- (1-170) and GST⅐SENP2-(1-70)) specifically retained a mAb414-reactive band that migrated with a mobility corresponding to 180 kDa on gel electrophoresis. Previous characterization of mAb414-reactive proteins in Xenopus egg extracts (15) suggested that the 180-kDa band was likely to be the nucleoporin Nup153 (16). To confirm this identification, we subjected the same samples to Western blotting using antibodies against the Xenopus Nup153 (17).…”

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

“…It has been shown that in vitro nuclear assembly spontaneously in Xenopus egg interphase extracts a double nuclear membrane with nuclear pore forms around added chromatin, whether natural animal or plant sperm chromatin or exogenously added procaryotic DNA is used [3][4][5]. Some information concerning reassembly of nuclear membranes, organelles, and individual nucleosomes has arisen from the use of cell-free extracts derived from eggs of Xenopus or sea urchin, and somatic cells and embryos of Drosophila or Nicotiana [2,[6][7][8][9][10][11][12][13][14].…”