Biochemical characterization of nuclear pore complex protein gp210 oligomers.

The membrane-spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N-terminal domain, a short C-terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration. Cross-linking of proteins in isolated membranes prior to solubilization dramatically increases the proportion of dimers. The dimers are SDS-resistant, as previously observed for some integral membrane proteins of cis-Golgi and plasma membrane proteins, including glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration and denaturing electrophoresis, but unlike the dimers are dissociated by reduction and heating in the presence of SDS. We propose that gp210 is organized into the pore membrane as a large array of gp210 dimers that may constitute a luminal submembranous protein skeleton.     

Dynamic properties of nuclear pore complex proteins in gp210 deficient cells

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.     

Oxalate-induced and cell-cycle-dependent expression of nuclear pore complex oxalate binding protein gp210

The effect of oxalate, a constituent of renal stone, on the expression of nuclear pore complex oxalate binding protein (gp210) in Vero monkey kidney cells was examined. The expression of this protein was found to increase more in mitotic phase than in S phase, suggesting cell cycle dependency. Exposure of cells to oxalate-containing growth medium resulted in a relative increase in nuclear pore complex oxalate binding protein in each stage of cell cycle. The concentration of this protein was found to increase six times in the telophase stage of the cells exposed to high concentrations of oxalate in the growth medium, though slight reduction in cell density was observed. Structural analogues of oxalate did not show any stimulatory effect on expression of this oxalate binding protein. Hence, the expression of the nuclear pore complex oxalate binding protein gp210 was specific to oxalate and is cell cycle dependent.     

Polyproline type II conformation in the C-terminal domain of the nuclear pore complex protein gp210

gp210 is a major constituent of the nuclear pore complex (NPC) with possible structural and regulatory roles. It interacts with components of the NPC via its C-terminal domain (CTD), which follows a transmembrane domain and a massive ( approximately 200 kDa) N-terminal region that resides in the lumen of the perinuclear space. Here, we report the solution structure of the human gp210 CTD as determined by various spectroscopic techniques. In water, the CTD adopts an extended, largely unordered conformation, which contains a significant amount of left-handed polyproline type II (PII) helical structure. The conformation of the CTD is altered by high pH, charged detergents, and the hydrogen bond-promoting reagent trifluoroethanol (TFE), which decrease the PII fraction of the fragment. TFE also induces a conformational change in a region containing an SPXX motif whose serine becomes specifically phosphorylated during mitosis. We propose that PII elements in the CTD may play a role in its interaction with the NPC and may serve as recognition sites for regulatory proteins bearing WW or other, unknown PII-binding motifs.     

Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex

The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo. An alanine substitution mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. Our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC.     

The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.     

A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.

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.     

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.     

Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail

Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC(50)s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread.     

PDZ domain protein GIPC interacts with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-related protein-1).

Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation. Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains. To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library. Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein. The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein. Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC. Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state. Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region. Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes.     

A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors

An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass (CPG) beads. Soluble receptor--3H-steroid complex (cytosol or nuclear extract) is adsorbed quantitatively within the crevasses of porous glass beads. Excess labeled steroid as well as most non-specifically bound steroid is easily washed away, leaving the hormone-receptor complex retained by the beads. Bound 3H-steroid is eluted with ethanol and measured for radioactivity. This procedure which is simple, rapid, and highly reproducible is carried out using frozen samples (stable for many months) containing as few as 1 X 10(7) cells. A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration, while the CPG and whole cell assays provide equivalent values for total receptor content.     

Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.     

Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes

The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.     

Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion.     

Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement

The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail- or FDelta19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail- mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail- mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail- is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.     

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.     

Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.