Monoclonal antibodies to a Mr 68000 pore complex glycoprotein interfere with nuclear protein uptake in Xenopus oocytes

Abstract

Using a monoclonal antibody (PI1) raised against mouse lymphocyte nuclear matrix fractions we have identified a N-acetylglucosamine (GlcNAc)-containing glycoprotein of Mr 68,000 as a component of the nuclear pore complexes of Xenopus laevis oocytes. The antigenic determinant recognized by antibody PI1 comprises both the sugar moiety and protein sequences since, on the one hand, added GlcNAc competed effectively for antibody binding and, on the other hand, the antibody reacted in immunoblots with only one member of the GlcNAc-containing pore complex glycoprotein family. By using immunogold-electron microscopy we could demonstrate that the Mr 68,000 glycoprotein was located preferentially to the cytoplasmic side of the pore complex channel. When radiolabeled soluble nuclear proteins were injected into the cytoplasm of Xenopus oocytes, their reentry into the nucleus was almost completely inhibited in the presence of antibody PI1 as shown by two-dimensional gel electrophoresis. The results indicate that the evolutionarily conserved Mr 68,000 glycoprotein is involved in transport processes of karyophilic proteins from the cytoplasm into the nucleus.

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

“…, 1986). lt recognizes a major GlcNAccontaining pore complex glycoprotein of Mr 68,000 both from XC/lOPUS oocytes (Dabauvalle et al 1988b) and mammalian PtK2 cells (Benavente et al 1989 a). PlI antibodies purificd from ascites fluid by hydroxylapatite chromatography (Stanker et al 1985) werc kindly provided by N. Chaly (Ca riet on University, üttawa.…”

“…7, lane 4, arrows) should considerably exceed a molecular mass 01' 254,000. We surmise that antibody PI1 also binds, under the conditions 01' immunoadsorption, other glycoproteins not associated with p68 since the GlcNAc sugar moiety forms part ofthe immunological determinant (Dabauvalle et al 1988b). In order to identify unequivocally the protein(s) associated with p68 and to clarify wh ether they are also constituents 01' pore complexes it will be necessary to purify the p68 complexes as far as possible prior to immunoprecipitation.…”

“…Since antibody Pli has previously only been characterized by immunoblotting on isolated nuclear envelopes (Dabauvalle et al 1988b), we first performed experiments to verify that it recognized the same antigen in Xenopus egg extract. Total proteins of extracts from activated and non-activated eggs were separated by gel electrophoresis, bIotted and probed with antibody Pl1 .…”

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

“…Experiments were carried out using PIl ascites fluid purified by hydroxylapatite chromatography, concentrated and lyophilized as previously described (Stanker et al 1985). In Xenopus oocytes it has been shown by protein blotting and immunogold electron microscopy that the reconstituted lyophilized PI1 column fraction recognizes specifically a major Mr 68000 glycoprotein located in the pore complex channel (Dabauvalle et al 1988a). Guinea pig antibodies against nucleoplasmin were kindly provided by Georg Krohne (German Cancer Research Center, Heidelberg, FRG; see Krohne 1985).…”

“…As shwon in Figure 2, the antibody reacted specifically with a single polypeptide of Mr 68000 (lane 2', arrow). A poly- peptide band of identical Mr value has been detected with PI1 in immunoblots using isolated nuclear envelopes from Xenopus oocytes (Dabauvalle et al 1988a) and of slightly higher electrophoretic mobility when bovine lymphocyte nuclear fractions were used as the antigen source (Chaly et al 1984).…”