Abstract. Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonaI antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.T HE nuclear envelope forms the membrane boundary of the nucleus in eukaryotic cells (for reviews see references 13 and 14). Its principal functions include compartmentalization of nuclear metabolism and control of selective macromolecular movement between nucleus and cytoplasm. The major structural components of the nuclear envelope are inner and outer nuclear membranes, nuclear lamina, and pore complexes. The nuclear lamina (reviewed in references 16 and 33) is a meshwork of intermediate filaments lining the nucleoplasmic surface of the inner nuclear membrane, which is thought to provide a framework for the regulation of nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromosomes (18). Pore complexes constitute large supramolecular structures present at regions where inner and outer nuclear membranes are joined to form pores (2, 15), and are thought to be the major or exclusive sites of molecular exchanges across the nuclear envelope (11,13,14,39).Based on cell microinjection studies, the pore complex appears to contain an aqueous channel of ,,o10 nm in diameter through which solutes and sufficiently small macromolecules (e.g., proteins <20-40 kD) can migrate by passive diffusion (35,41). However, nncleocytoplasmic transport of larger macromolecules probably involves mediated mechanisms (7, 10). Recent work has shown that certain nuclear proteins contain discrete structural regions (6) or sequences (2...

Abstract. A novel form of protein-saccharide linkage consisting of single N-acetylglucosamine (GIcNAc) residues attached in O-linkages directly to the polypeptide backbone has been described (Holt, G. D., and G. W. Hart, 1986, J. Biol. Chem., 261:8049-8057). This modification was found on proteins distributed throughout the cell, although proteins bearing O-linked GIcNAc moieties were particularly abundant in the cytosolic and nuclear envelope fractions of rat liver. In the accompanying article (Snow, C. M., A. Senior, and L. Gerace, 1987, J. Cell. Biol., 104: 1143-1156, the authors describe monoclonal antibodies directed against eight proteins localized to the nuclear pore complex. These proteins occur on the cytoplasmic and nucleoplasmic (but not lumenal) sides of nuclear membranes. In this report, we demonstrate that all members of this group of pore complex proteins bear multiple O-linked GIcNAc residues. Further, we show that the O-linked GIcNAc moieties are linked via serine (and possibly threonine) side chains to these proteins. Perturbing the O-linked GIcNAc residues either by covalently attaching galactose to them or by releasing them with 13-N-acetylglucosaminidase strongly diminishes the immunoreactivity of the proteins with all of the monoclonal antibodies. However, the O-linked GIcNAc moieties are only part of the epitopes recognized, since O-GlcNAc-containing limit pronase fragments of nuclear pore complex proteins cannot be immunoprecipitated by these antibodies. These findings, taken together with those in the accompanying article, are a direct demonstration that proteins of the cytoplasm and nucleoplasm bear O-linked GIcNAc residues. p REVAILING evidence suggests that the bulk of the wellstudied types of carbohydrate moieties on glycoconjugates are either localized to the cell surface or within lumenal compartments of intracellular organelles (e.g., lysosomes, Golgi, and endoplasmic reticulum). In contrast, relatively little evidence has been obtained suggesting the existence of glycoproteins in the cytoplasmic and nucleoplasmic compartments of cells. Early studies involving carbohydrate analyses of nuclei and nuclear matrix fractions indicated the presence of glycoconjugates in these fractions (reviewed in Furukawa and Terayanm, 1979;Stein et al., 1981;Stoddart, 1979), and the presence of cytoplasmic glycoproteins has also been suggested (Meyer and Burger, 1976). These data have received support in several more recent investigations (e.g., Kan and Pinto da Silva, 1986;Fedarko and Conrad, 1986;Nagakura et al., 1986). However, attempts to identify and characterize nucleoplasmic and cytoplasmic glycoproteins have been severely hampered by cross-contamination with other cellular compartments, and detailed biochemical analyses of most putative cytoplasmic and nucleoplasmic glycoproteins is lacking.Recently the subcellular distribution of glycoproteins bearing terminal N-acetylglucosamine (GIcNAc) ~ residues was surveyed (Holt and Hart, 1986) by employing galactosyltransferase, an enzyme that trans...

Abstract. We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis. T HE nuclear envelope forms the boundary of the nuclear compartment in eukaryotes (reviewed in references 10, 13, 25). Its primary functions involve selective transport of macromolecules between the nucleus and the cytoplasm and organization of higher level nuclear architecture. The major components of the nuclear envelope are inner and outer membranes, nuclear pore complexes and the nuclear lamina. Nuclear pore complexes, which occur at regions where the inner and outer membranes are joined to form pores, are large supramolecular assemblies that provide channels for molecular movement across the nuclear envelope (30). The outer nuclear membrane is morphologically continuous with the rough and smooth endoplasmic reticulum (ER) I and has functional properties of ER, while the inner nuclear membrane is lined by a filamentous meshwork called the nuclear lamina. In higher eukaryotic ceils, the lamina contains mainly a polymer of one to four related proteins (nuclear lamins) which are biochemically and structurally very similar to cytoplasmic intermediate filament proteins (9, 11). The lamina is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromosomes (11). During mitosis in higher eukaryotic cells the A. Senior's present address is

One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water‐soluble fragment of gp210 containing concanavalin A‐reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti‐peptide antibodies against two sequences near the C‐terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane‐spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.