Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Abstract

Closed nuclear envelope ghosts in the physiological orientation were prepared from rat liver nuclei as previously described. Here we report transport measurements of various proteins and ribonucleic acids across the envelope Qf these vesicles. Histones were accumulated rapidly in the ghosts, in contrast to other, nonnuclear, proteins. Triton X-100 removal of the external nuclear membrane from loaded vesicles, as well as comparative studies with open vesicles, excluded the effects of external adsorption. The exchange rate of histones across the nuclear envelope is strongly depressed in the presence of GTP and GDP. The vesicles contain the translocation mechanism for poly(A)-containing RNA. The translocation of poly(A), messenger RNA, and ribosomal RNA was investigated after entrapment of these nucleic acids during the preparation of vesicles. Our data show that the complete export of only poly(A)-containing RNA from the vesicles is enhanced in the presence of 2 mM ATP. This RNA, as well as poly(A), is transported unidirectionally.Previously, we described the preparation and characterization of closed envelopes from rat liver nuclei (1, 2). These vesicles were shown to be tight. The DNA content, calculated per mg of protein, is reduced to 1-1.5% of the value for whole nuclei.The high number of binding sites for nuclear constituents, such as histones or mRNA, renders the exact measurement of rates of migration into or out of whole nuclei difficult. Evidence from microinjection experiments (3,4), sequence analysis, and especially site-directed mutagenesis of proteins that accumulate in the nucleus (5, 6) has prompted the discussion of selectivity of nuclear membrane transport (7). The nuclear envelope ghosts seemed suitable for the further elucidation of these mechanisms.MATERIALS AND METHODS Leaky nuclear envelopes ("DNase envelopes") were prepared by hypotonic DNase I treatment as described by Dwyer and Blobel (8). The preparation of closed nuclear envelopes with heparin solutions was carried out as previously described (1, 2).Poly(adenylic acid) of an average molecular weight of 100,000 was purchased from Sigma, and the absence of poly(A) with molecular weight below 60,000 was verified by HPLC runs on a Nucleogen DEAE 500-10 column. NTPase activity was determined as described (1). Histones were prepared from calf thymus and fractionated by the procedure of Johns (9) (purity control, see Fig. 3 and ref. 10). Xenopus laevis eggs were obtained from the Max-Planck-Institute of Biophysics, Frankfurt, and nucleoplasmin was prepared from these as described (11). All proteins were labeled in 50 mM NaHCO3 by addition of [14C]acetic anhydride (Amersham Buchler), specific activity 120 mCi/mmol (1 Ci = 37 GBq). The proteins were then dialyzed against the buffer described in the legend of Fig. 3 Fig. 1, the closed vesicles still possess residual fringes of the chromatin material on the inside of the inner membrane; the pore complexes are revealed by freeze fracture. Phase-contrast light microscopy reveals a population...

Evidence for a direct interaction of Rev protein with nuclear envelope mRNA–translocation system

“…Previously we determined that the transport of RNA trapped in the vesicles is unidirectional and is promoted by ATP and the presence of a poly(A) sequence on the RNA [I31 (see also [53]). In these properties, efflux of RNA from the vesicles corresponds to nucleocytoplasmic transport of RNA in vivo.…”

In Vivo Kinetics of mRNA Splicing and Transport in Mammalian Cells

“…This could reflect both the excessive number of molecules injected and the importance of a proper localization of transcripts for their interaction with the transport machinery. Other experimental approaches include the use of nuclear envelope vesicles (36) and isolated nuclei (39). In the first case, it should be possible to investigate specifically the translocation of mRNA through the nuclear pore complex (NPC), provided a suitable environment is re-created.…”

Evidence for involvement of a nuclear envelope‐associated RNA helicase activity in nucleocytoplasmic RNA transport

“…After incubation (Riedel et al, 1987), the NE vesicles were prepared in 0.25 M-STKMCbuffer, as described above. Under these conditions, 0.2-0.6 ng of RNA was entrapped per mg of NE vesicles (based on protein content).…”