PROTEINS IMMUNOLOGICALLY RELATED TO DEHYDRINS IN FUCOID ALGAE

Adult fucoid algae on Atlantic shores have well-characterized, species-specific tolerances to the varying levels of desiccation that occur from the low to high intertidal zones; however, less is known about embryonic tolerances and their mechanistic basis. We investigated this by 1) exposing embryos of Fucus evanescens C. Agardh, F. spiralis L., and F. vesiculosus L. from the Maine shore to osmotic desiccation in hypersaline seawater and 2) examining whether these embryos contain species-specific dehydrins, proteins first identified in higher plants that are hypothesized to confer tolerance to dehydration. Embryonic survival when cultured in hypersaline seawater >100 practical salinity units (psu) correlated with the position of these species in the intertidal zone (F. spiralis > F. vesiculosus > F. evanescens), but all 1-day-old embryos of these species tolerated treatment with 100 psu or lower seawater. Proteins (17–105 kDa) immunologically related to dehydrins were detected on western blots with dehydrin antibodies raised against a synthetic peptide representing the conserved motif of dehydrins in higher plants. These proteins were constitutive and unstable when subjected to prolonged (>15 min) temperatures above 55° C, unlike most higher plant dehydrins, which are inducible and remain soluble at 75°–100° C. The presence of these proteins was species- and stage-specific. Sperm of F. vesiculosus had a characteristic protein of 76 kDa, whereas eggs and embryos (6 h to 3 days old) had a 92-kDa protein. By 1 week of age, expression of the 92-kDa protein decreased, and the 35-kDa protein of adults was present. Embryos of A. nodosum L. and Pelvetia compressa J. Agardh DeToni contained an 85-kDa protein rather than the 92-kDa protein of Fucus embryos (F. distichus L., F. evanescens, F. spiralis, and F. vesiculosus). The 92-kDa protein became more abundant in embryos exposed to hyperosmotic seawater at 50 psu (F. evanescens and F. vesiculosus) or 150 psu (F. spiralis); however, dehydrin-like proteins of some molecular masses decreased in abundance simultaneously. Further characterization of these proteins is required to establish whether they protect embryos against intertidal desiccation.     

The Spike Protein of Murine Coronavirus Mouse Hepatitis Virus Strain A59 Is Not Cleaved in Primary Glial Cells and Primary Hepatocytes

Mouse hepatitis virus strain A59 (MHV-A59) produces meningoencephalitis and severe hepatitis during acute infection. Infection of primary cells derived from the central nervous system (CNS) and liver was examined to analyze the interaction of virus with individual cell types derived from the two principal sites of viral replication in vivo. In glial cell cultures derived from C57BL/6 mice, MHV-A59 produces a productive but nonlytic infection, with no evidence of cell-to-cell fusion. In contrast, in continuously cultured cells, this virus produces a lytic infection with extensive formation of syncytia. The observation of few and delayed syncytia following MHV-A59 infection of hepatocytes more closely resembles infection of glial cells than that of continuously cultured cell lines. For MHV-A59, lack of syncytium formation correlates with lack of cleavage of the fusion glycoprotein, or spike (S) protein. The absence of cell-to-cell fusion following infection of both primary cell types prompted us to examine the cleavage of the spike protein. Cleavage of S protein was below the level of detection by Western blot analysis in MHV-A59-infected hepatocytes and glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. Thus, cleavage of the spike protein does not seem to be essential for entry and spread of the virus in vivo, as well as for replication in vitro.     

Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor

Abstract: His¹⁰¹ of the GABA_A receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [³H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [³H]Ro 15-4513 bound to all mutant receptors with equal or greater affinity than to the wild-type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15-1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15-1788 and Ro 15-4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.     

Human Immunodeficiency Virus (HIV) Envelope Binds to CXCR4 Independently of CD4, and Binding Can Be Enhanced by Interaction with Soluble CD4 or by HIV Envelope Deglycosylation

Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1_SF-2 gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1₄₅₁ bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did.In the recent past, several members of the family of chemokine receptors have been identified as cofactors for human immunodeficiency virus type 1 (HIV-1) entry (1, 6, 8, 10). Specifically, CCR5 (as well as CCR3 and CCR2b in some instances) has been shown to mediate entry of viruses characterized as macrophage tropic or dual tropic (1, 5–8), while CXCR4 has been shown to mediate entry of T-cell-tropic or dual-tropic strains (7, 10). While several ligands have been found for CCR5, CXC chemokine stromal derivative factor (SDF1) remains the only known ligand for CXCR4 (4, 24). Coimmunoprecipitation studies have shown that HIV-1 Env from T-cell-tropic strains forms a complex with CD4 and CXCR4 (18), but the nature of the binding events leading to the formation of this complex and the possibility of a direct interaction between HIV Env and CXCR4 remained speculative. Data from Hesselgesser et al. (15) have more recently shown that gp120 from the T-cell-tropic strains IIIB or BRU was able to compete with SDF1 for binding to CXCR4 in hNT cells (a neuronal CD4-negative cell line), indicating the possibility of a direct interaction between CXCR4 and gp120, but no information was presented on the relevance of the interaction with CD4. Other data have shown that gp120 from macrophage-tropic strains of HIV might be able to bind directly to CCR5 and that the affinity for binding between the two molecules can be increased significantly by the presence of soluble CD4 (sCD4) (34), although this effect could not be reproduced by a different group (32).We have performed the following studies to determine if HIV Env binds to CXCR4 independently of CD4 and, if so, what would be the effect of previous binding of HIV Env to sCD4.

CD4-independent binding of HIV Env to CXCR4.

The phenotypes of the T-cell lines CEM-SS and Jurkat 25 (J25) were evaluated with respect to surface expression of both CD4 and CXCR4. J25 clone 22F6 cells (3, 21) were grown in complete medium (RPMI 1640, 2% penicillin-streptomycin, 2% l-glutamine; BioWhittaker, Walkersville, Md.) containing heat-inactivated 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. CEM-SS is a T-cell line that was obtained from the AIDS Research and Reference Reagent Program and maintained in complete medium. CEM-SS cells were derived from a human lymphoblastoid tumor (22, 23). Commercial monoclonal antibody (MAb) to CD4 (mouse immunoglobulin G2a [IgG2a], clone S3.5), fluorescein isothiocyanate (FITC) labeled, and the necessary isotypic controls were obtained from Caltag Laboratories (San Francisco, Calif.). Mouse MAb 12G5 against CXCR4 was raised in BALB/c mice and has been described previously (9). Goat anti-mouse IgG–FITC was purchased from Becton Dickinson (San Jose, Calif.). Flow cytometric analysis was performed on a Becton Dickinson FACScan cytometer equipped with a 15-mW argon laser emitting at 488 nm. Dead cells were detected on the basis of their scatter and eliminated from the analysis. Live cells (10,000) were analyzed for each marker. CXCR4 surface expression was determined by washing the cells taken in logarithmic growth phase with phosphate-buffered saline (PBS) containing 1% horse serum and incubating them with 10 μl of 12G5 antibody/100 μl (0.16 mg/ml) at 4°C for 30 min. The cells were then washed again in PBS, and a secondary goat anti-mouse IgG–FITC (Becton Dickinson) was incubated with the cells for another 30 min at 4°C. Finally, the cells were washed with PBS and fixed with 2% paraformaldehyde. As a control, equal amounts of mouse IgG2a (the same isotype as 12G5) were used. Both cell lines expressed significant levels of CXCR4 on their surfaces (Fig. ​(Fig.1),1), but only CEM-SS had measurable levels of surface CD4. This characteristic of the phenotype of J25 cells, with respect to CD4 expression, has been reported before (3). To assess binding of HIV Env to CXCR4, the following binding assay was developed. Oligomeric gp160 (ogp160) was purified from cell cultures (obtained from T. C. Van Cott (Henry M. Jackson Foundation, Rockville, Md.) infected with HIV₄₅₁ (17). The cells were washed once with PBS and then incubated with ogp160 for 1 h at 37°C in RPMI medium. The cells were washed again in PBS and incubated with 10 μg of human MAb 1331A [IgG3(λ)]/ml, which is specific for the C terminus of gp120 (i.e., amino acids 510 to 516 of HIV_LAI), or with a human MAb against p24 (MAb 71-31) as a control (12) for 30 min at 4°C. The secondary antibody was a goat anti-human IgG phycoerythrin labeled (Caltag). The cells were fixed in 2% paraformaldehyde, and the fluorescence intensity was determined by flow cytometry. Background was obtained by adding MAb 1331 and goat anti-human IgG, phycoerythrin labeled, to the cells in the absence of ogp160. The results of the binding assay with ogp160 from HIV₄₅₁ and both cell lines are shown in Fig. ​Fig.2A.2A. By using the high-affinity human MAb 1331A against the C-terminal region of gp120, our assay was able to detect significant binding of the ogp160 molecule to the surfaces of both cell lines even at concentrations of only 88 nM. The very high relative affinity of MAb 1331A for the gp120 molecule appears to be critical to demonstrate this interaction, as other antibodies with lower relative affinities for gp120 were incapable of detecting this low-level binding (data not shown). The binding of ogp160 to the CD4-expressing CEM-SS cells was several orders of magnitude higher than that to the J25 cells. To prove the specificity of the binding assay for CXCR4, a synthetic form of SDF1 was produced and tested for its ability to block infection by the HIV-1 strain NL_4-3 in HeLa CD4-positive long terminal repeat (LTR)-LacZ cells. These data have been published elsewhere (2). SDF1 synthesis and composition have been described previously (24). Exposure of J25 cells to SDF1 was shown to produce a dose-dependent blockage of the binding of ogp160 to the surfaces of the J25 cells (Fig. ​(Fig.2B),2B), indicating the specific nature of the assay. Open in a separate windowFIG. 1Phenotype analysis of CEM-SS and J25 cell lines. Thin solid line, background; thick solid line, CD4; dashed line, CXCR4.Open in a separate windowFIG. 2(A) Binding of ogp160 from HIV₄₅₁ to the surfaces of CEM-SS or J25 cells. Fluorescence intensity is expressed on a logarithmic scale on the x axis, with each line representing one-half log. Concentrations of ogp160 are shown at the right of each graph. The experiments were done in duplicate to ensure consistency of results. (B) Effect of RANTES (250 nM) or increasing amounts of SDF1 (up to 250 nM) on binding of ogp160 (355 nM) to J25 cells. The results are expressed as mean channel fluorescence. Experiments were repeated twice to ensure consistency of results.To further test the fact that HIV Env binding to CXCR4 could occur independently of CD4, and to evaluate the effect of prior binding of Env to sCD4, the following experiments were performed. We preexposed CEM-SS as well as J25 cells to either the anti-CD4 antibody Leu3a (Becton Dickinson), which blocks the CD4 binding domain of HIV Env, or OKT4 (Ortho Diagnostics, Costa Mesa, Calif.), which does not block binding of HIV Env to CD4. The cells were then tested for their ability to bind ogp160 to their surfaces. As shown in Fig. ​Fig.3,3, OKT4 had no significant effect on the binding of ogp160 to either CEM-SS or J25 cells while Leu3a readily inhibited binding of ogp160 to CEM-SS cells but had no such effect on J25 cells. Furthermore, when ogp160 was allowed to react in advance with recombinant sCD4 produced in CHO cells (Intracel, Issaquah, Wash.) for 30 min at 4°C at a concentration of 1 μg/ml, we were able to show a clear decrease in the surface binding of ogp160 to CEM-SS cells while the opposite, an obvious enhancement in surface binding, was demonstrated for J25 cells (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ogp160 to CEM-SS or J25 cells after exposure of the cells to the anti-CD4 antibodies Leu3a (thin solid lines), OKT4 (dotted lines), or a combination of ogp160 with sCD4 (dashed lines). The shaded areas represent background. The thick solid lines represent binding in the absence of antibodies or sCD4. The experiments were performed in quadruplicate with similar results. Mean channel fluorescence is represented on the x axis.Taken together, these data indicate that HIV Env can bind to CXCR4 independently of CD4. On the other hand, prior interaction of HIV Env with CD4 results in a clear increase in the binding of HIV Env to CXCR4.

Relevance of the glycosylation state of HIV Env in binding to CXCR4.

The binding of HIV Env to CD4 is dependent on the appropriate conformation of the Env molecule (27), which can be significantly altered by changes in its carbohydrate content. We next tested the hypothesis that alterations in the carbohydrate moieties of Env would affect its binding to CXCR4. To do so, we used the gp120 molecule from HIV_SF2, produced in CHO cells, and its counterpart, nonglycosylated HIV_SF2 Env 2-3, produced in yeast strain 2150, and tested both in the binding assay with CEM-SS or J25 cells. HIV_SF-2 gp120 and its nonglycosylated counterpart, Env 2-3, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Kathelyn Steimer, Chiron Corp. (13, 14, 19, 26, 29–31). The results are shown in Fig. ​Fig.4.4. As expected, nonglycosylated HIV_SF2 Env 2-3 bound to the surfaces of the CEM-SS cells to a lesser extent than did HIV_SF2 gp120. On the other hand, and unexpectedly, nonglycosylated HIV_SF2 Env 2-3 bound much more readily to the surfaces of the J25 cells than its glycosylated counterpart, HIV_SF-2 gp120, even when used at equal molar concentrations. To determine whether these findings could be generalized to other Env molecules that lacked intact carbohydrate molecules, we treated ogp160 with sodium metaperiodate. ogp160 from HIV₄₅₁ at 1.25 μg/ml was treated with sodium metaperiodate (Sigma, St. Louis, Mo.) in acetate buffer for 2 h at 4°C in the dark (33). The cells to be tested had been treated previously with 1% glycine (Sigma) for 30 min at 37°C. Such treatment results in the oxidation and cleavage of the carbohydrate hydroxyl groups without affecting the structure of the polypeptide chains (33). Nonspecific binding by the resulting aldehyde groups was prevented by blocking the target cells beforehand with 1% glycine. The results are shown in Fig. ​Fig.4.4. Sodium metaperiodate treatment of ogp160 resulted in a marked inhibition of the binding of ogp160 to the surfaces of the CEM-SS cells. In contrast, sodium metaperiodate treatment of ogp160 resulted in a very clear increase in the binding of HIV Env to the surfaces of the J25 cells. The preexposure of CEM-SS cells to SDF1 did not significantly affect the binding of ogp160 or sodium metaperiodate-treated ogp160. On the other hand, preexposure of J25 cells to 250 nM SDF1 resulted in a marked decrease in binding of both ogp160 and sodium metaperiodate-treated ogp160. These data indicate the specificity of the interaction of the deglycosylated form of ogp160 with CXCR4. The results of these experiments suggest that the alteration in the carbohydrate content of the HIV Env molecules resulted in a better exposure of the epitopes involved in gp120 binding to CXCR4. Open in a separate windowFIG. 4Binding of HIV_SF-2 gp120 or the nonglycosylated form, HIV_SF-2 Env 2-3 (Non-glyc SF-2 gp120), to CEM-SS or J25 cells. The concentration was 355 nM for both. The binding of ogp160 and sodium metaperiodate-treated ogp160 (De-glyc ogp160), each at a concentration of 355 nM, to CEM-SS or J25 cells is also shown. The two right-hand bars in each graph show results for cells preexposed to SDF1 at 150 nM. The results are expressed as mean channel fluorescence. The experiments were performed in duplicate with similar results.The understanding of the underlying mechanisms by which HIV Env, CD4, and the newly discovered HIV coreceptors interact to mediate viral entry remains a very significant issue. The way that HIV Env and CD4 interact is well established (28), and some information exists about the interaction between HIV Env, CCR5, and CD4 (34). In this paper we have shown that HIV Env is able to interact in a CD4-independent manner with CXCR4. Still, the extent of such interaction was clearly lower than that of the sCD4-HIV Env complex and CXCR4. This effect of sCD4 seems to be consistent with the observation that the complexing of this molecule with HIV Env from the strains JRFL or BAL resulted in a significant increase in the affinity of HIV Env for CCR5 (34). We speculate that this interaction between sCD4 and HIV Env results in a conformational change that exposes the binding epitopes in HIV Env relevant for binding to CXCR4, as it does with other gp120 epitopes (16). A different scenario would involve a change in both molecules, resulting in a newly formed common binding epitope. This second alternative seems less likely given our data showing CD4-independent binding of HIV Env to CXCR4, as well as previous data showing the existence of HIV strains capable of CD4-independent entry into target cells (9, 15).The gp120 molecule from HIV contains 20 potential N-linked glycosylation sites, with N-linked glycans representing at least 50% of the molecular mass. Their role in CD4 binding has been studied extensively, although some of the results remain somewhat controversial. Most of the available data seem to indicate that complete lack of glycosylation completely (20), or at least partially (25), inhibits HIV Env binding to CD4. Also, enzymatic manipulation of the carbohydrate residues results in a significant decrease but not in complete abrogation of the binding of HIV Env to CD4 (11, 20, 25). It was therefore somewhat unexpected to find that the nonglycosylated form, as well as the sodium metaperiodate-treated form, of HIV Env was able to bind in such an enhanced way to CXCR4. This would appear to reinforce the concept of the existence of a binding epitope for CXCR4 within HIV Env which is different from the one for CD4. It also suggests that the changes occurring as a consequence of the manipulation of the carbohydrate residues likely result in a better exposure of the CXCR4 binding epitope(s) within the HIV Env molecule.In summary, we have shown that HIV Env can interact with CXCR4 in a CD4-independent manner. We have also shown how the interaction of CD4 with HIV Env results in a significant increase in the binding of the latter to CXCR4 and how the alterations in the carbohydrate composition of the HIV Env molecule affect its binding to CXCR4. The complete definition of these interactions may result in novel approaches to protect against cell infection by HIV.     

A case report of a male rank reversal in a group of wild white-faced capuchins (Cebus capucinus)

During the course of a study of social relationships in wild, white-faced capuchins at Lomas Barbudal, Costa Rice (May 1990–May 1993), the alpha male was deposed by a subordinate male. The rank reversal was preceded by a decline in proximity maintenance by females to the alpha male, and an increase, in the amount of aggression directed toward the alpha male by the beta female and her female coalition partners. At the time of the rank reversal, females switched from giving thegargle vocalization exclusively to the old alpha male to gargling to the new alpha male; however, juveniles were less consistent with regard to which male they gargled to. At the time of the rank reversal, most adult females reduced the time spent in proximity and grooming with the old alpha male, and increased the time spent in proximity and grooming with the new alpha male. In contrast, juveniles' patterns of affiliation with males did not change in a predictable way following the reversal. The social strategies employed by capuchin monkeys during this rank reversal are compared with those of chimpanzees.     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

The Saccharomyces cerevisiae Prenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.     

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57p^E17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.     

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.