Abstract
Nuclear pore proteins bearing O-linked N-acetylglucosamine (GlcNAc) are involved in nuclear transport, although a role for their glycosylation is not established. Xenopus egg extracts capable of reforming functional nuclei in vitro yielded nuclei with impaired transport and reduced nuclear pore density when depleted of wheat germ agglutinin-binding proteins. Many of the nuclear pores remaining in wheat germ agglutinin-depleted nuclei showed a striking loss of internal structure. Nuclear transport and normal nuclear pore structure were restored by the addition of nuclear pore glycoproteins from rat liver or Xenopus eggs. Glycoproteins modified by the addition of galactose to O-linked GlcNAc were also competent for assembling normal nuclear pores and restoring nuclear transport. Aphidicolin-sensitive DNA synthesis was unaffected by the removal or modification of O-linked GlcNAc glycoproteins. These data argue against a requirement for a lectin-like recognition of O-linked GlcNAc glycoproteins in nuclear pore assembly, nuclear transport, or DNA synthesis.
Highlights
From the Laboratory of Biochemistry and Metabolism, National Instituteof Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Many of the poorly understood, p62 and the other 0-linked glycosylation examined cosamine (GlcNAc) pore nuclear pores remaining in wheat germ agglutinin-de- proteins have been shown to be involved in nuclear transport pleted nuclei showed a striking loss of internal struc- based on the observations that: (i) WGA,l a GlcNAc-specific ttgtGnuueluylirrcncceeNlwso.eApaeNmrrcrwueoorpctedeelroesiienfrtaioesrersdeftabrdarlobanysmoyndttcshhoprreemeoaasradtpttddoeldiaritvtiieninteonidrgnotonfnoonfXnrorouuarefcmncslgsleoeeaaapamllruarpnscbtoturleoiracgnselngegessant.proooGr0prmlt-oy.lraicAneol kppserthrodiu-dci--(lmFpeioiicen)rytltdieanieryn,pialnevenihttdtiiarobolniF.t(,soDonr1fbau90bec9-asl1le,ui1)anv9;rka(8tierl8ilad;ien)WeGsntpoulacolcfNtrfl.tee,A(ot1Fcpa9iong9lr.ll0,iyan;1ycFs9oi8pinn8rlo;taeYtyeeroiatnnacasentlddiw.naF,iheot1ihtrb9b8aiect7lssy.;,t1toN1r9sa9e8on9w7lsi0)c--;; Colin-sensitiveDNA synthesis was unaffected by the re- factors required for nucleartransport(Sterne-Marr et a l . , movalor modification of0-linkedGlcNAc glycoproteins. 1992)
Previous work has shown that the majority of nuclear pore glycoproteins reside in the cytosolic fraction of the extract and canbe efficiently depleted from the cytosol using WGA lectin affinity chromatography (Finlay and Forbes, 1990; Finlay et al, 1991).In the current studies, removal of the glycoproteins from the cytosol was glycoproteins
Summary
Sion of membranes, 0.5-volume of 6 mg/ml creatine phosphokinase, 1 volume of 20 x energy mixture UDP-[14C]Gal(>200 mCiimmo1) for 15 min a t 37 "C before the addition Fluorescence Microscopy-Aliquots of each reformation mixture were of unlabeled UDP-Gal. One volume of r a t nuclear supernatant was fixed at 4 "C with either 1m 0 ethylene glycol succinimidylsuccinateor incubated 1-2 h with a 0.5 volume of either packed WGA or ECL- 5% formalin (froma 10% stock madein buffer D). SDS-PAGEa n d Protein Blotting-Aliquots of affinity-purified glyco- GlcNAc glycoproteins from Xenopus or rat has been shown to proteins and reformation mixtures were analyzed on 10% SDS-PAGE and either blotted onto nitrocelluloseor prepared for autoradiography using Enlightning accordintgo the protocols of the manufacturersL. Immunoblots were incubated first with diluted anti-p62 antirestoretheabilityto reform functional nuclei (Finlayand Forbes, 1990;Finlay etal., 1991).For these studiews,e used rat nuclear glycoproteins, because the major nuclear pore glycoprotein from rat, p62, can be readily distinguished from the Xenopus p62 using rat p62-specific antisera. This allowed direct monitoring of the modification and incorporation of exogenously added nuclear pore
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