A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.

The cutaneous T cell lymphoma, mycosis fungoides, is a human T cell lymphotropic virus-associated disease. A study of 50 patients.

For nearly two decades it has been suspected that the cutaneous T cell lymphoma, mycosis fungoides (MF), and its leukemic variant, the Sézary syndrome, are caused by the human T lymphotropic virus (HTLV-I/II). Arguments against this concept included the finding that only a small number of MF patients have antibodies to HTLV-I/II and that attempts to detect proviral sequences by mere Southern hybridization of extracted DNA usually met with failure. However, we have reported repeatedly that HTLV-like particles emerge in blood mononuclear cell (PBMC) cultures of practically all patients with this disease. In several instances, the particles were identified as HTLV by immunoelectron microscopy as well as biomolecular analysis. With the assumptions that the virus in MF patients may have become detection by Southern hybridization alone, the extracts of freshly isolated PBMC of 50 consecutive patients were subjected to combined PCR/Southern analysis. Here we report the presence of HTLV pol and/or tax proviral sequences in 46 out of 50 (92%) of the patients tested. In addition, five of the patients, who lacked antibodies to HTLV-I/II structural proteins, were found to be seropositive for tax. It thus seems reasonable to conclude that MF/Sézary syndrome is an HTLV-associated disease and that lack of an immune response does not preclude infection with this type of virus.

Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line.

A mixed-oligonucleotide probe was used to identify four ras-like coding sequences in a human teratocarcinoma cDNA library. Two of these sequences resembled the rho genes, one was closely related to H-, K-, and N-ras, and one shared only the four sequence domains that define the ras gene superfamily. Homologs of the four genes were found in genomic DNA from a variety of mammals and from chicken. The genes were transcriptionally active in a range of human cell types.

Effects of mutant Ran/TC4 proteins on cell cycle progression.

Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.

Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Identification and characterization of a human homolog of the Schizosaccharomyces pombe ras-like gene YPT-3.

The Polymerase Chain Reaction was used to amplify ras and ras-like sequences from two human cDNA libraries. Members corresponding to each of the three major ras-subfamilies (ras, rho, and rab/YPT) were identified. The one homologous to rab/YPT, referred to here as YL8, appears to be the human homolog of the recently reported Schizosaccharomyces pombe YPT3 gene. The YL8 gene could encode a guanine nucleotide binding protein of 216 amino acids with about 70% amino acid sequence identity to S. pombe YPT3, and is transcriptionally active in a variety of human cell lines.

Evolutionary implications of primate endogenous retroviruses.

Endogenous DNA sequences related to retroviruses are probably present in all primates. By using approaches based on the polymerase chain reaction, two separate studies have revealed the evolutionary history of some of these sequences. In the first study, a retrovirus-like reverse transcriptase (RT) sequence homologous to that of Baboon endogenous virus (BaEV) has been identified in both Old World monkeys and African apes, but not in humans or Asian apes. This RT sequence is highly conserved at the amino acid level, but not the nucleotide level, in the baboon, African green monkey, Java macaque, chimpanzee, and gorilla. The patterns of nucleotide substitution indicate functional conservation and suggest that this RT sequence was present in the primate germline before apes and Old World monkeys diverged about 30 million years ago. In the second study, a comparison of endogenous proviral DNAs and their adjacent sequences has been used to analyze the evolutionary history of three previously reported human endogenous retroviruses, HERV-E(4.14), HERV-R(3), and HERV-Ia. It is shown that these retroviruses have also been resident in the primate line since before the ape-Old World monkey divergence. The implications of the presence of functionally conserved RT genes in the germlines of primates, and the potential for using integration sites as tools for analyzing phylogenetic relationships among primates and their retroviruses, are discussed.

Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.

The human Ras-related nuclear protein Ran/TC4 (refs 1-4) is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1 (refs 12, 13), interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, we searched for a Ran-specific GTPase-activating protein (GAP), and for putative effectors (proteins that interact specifically with Ran/TC4-GTP). We report here the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran/TC4-GTP.

Kinetics of protein import into isolated Xenopus oocyte nuclei.

An in vitro assay for nucleocytoplasmic transport was established in which signal-dependent protein import is reproduced faithfully by isolated purified nuclei. The assay permits the precise quantification of import kinetics and the discrimination between translocation through the nuclear envelope and intranuclear transport. Nuclei were manually isolated from Xenopus oocytes and after manual purification incubated with a medium containing a green fluorescent transport substrate, karyopherins alpha2 and beta1, a red fluorescent control substrate, an energy mix and, for keeping an osmotic balance, 20% (wt/vol) BSA. Import of transport substrates into the nucleus and exclusion of the control substrate were monitored simultaneously by two-color confocal microscopy. Two widely differing import substrates were used: the recombinant protein P4K [480 kDa, four nuclear localization sequences (NLSs) per P4K tetramer], and NLS-BSA (90 kDa, 15 NLSs). The measurements suggested that import, at the specific conditions used in this study, consisted of two consecutive processes: (i) the rapid equilibration of the concentration difference across the nuclear envelope, a process involving binding and translocation of substrate by the nuclear pore complex, and (ii) the dissipation of the intranuclear concentration difference by diffusion.

Detection of human T-lymphotropic virus-like particles in cultures of peripheral blood lymphocytes from patients with mycosis fungoides.

Because of seronegativity and absence of a leukemic phase in most patients with mycosis fungoides, a role for the human T-lymphotropic virus type I (HTLV-I) in this disease has remained tenuous. Virus particles are not seen in fresh isolates of skin or blood lymphocytes and the malignant cells (Sézary cells) have been difficult to culture. The availability of growth factors and biomolecular techniques have prompted a renewed attempt to find evidence of virus infection in these patients. We report here the successful culture of blood lymphocytes of 17 patients with mycosis fungoides and 1 patient with the Sézary syndrome. The cells of 2 additional patients failed to grow after 4-6 weeks in vitro. Ultrastructural analysis of the cultures showed an abundance of HTLV-like particles in the specimens of 18 of the 20 patients. Preliminary immunohistochemical studies carried out with various antisera directed against HTLV-I and the polymerase chain reaction utilizing a probe for a conserved region of the pol gene of HTLV-I were positive on only a portion of the specimens. Although definitive characterization of this organism awaits further analysis, it seems likely that circulating lymphocytes of all patients with mycosis fungoides harbor a virus that morphologically resembles HTLV-I.

Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region.

The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell lambda expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.

Tissue-specific expression of Ran isoforms in the mouse.

Ran genes encode a family of well-conserve small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. We have now characterized patterns of Ran gene expression in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies.

Leishmania species: mechanisms of complement activation by five strains of promastigotes.

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.

HPV-16-related DNA sequences in Kaposi's sarcoma.

In the USA, Kaposi's sarcoma associated with the acquired immunodeficiency syndrome (AIDS-KS) is ten times more common in homosexual or bisexual men than in heterosexual men with AIDS. One explanation for this finding is that AIDS-KS may be caused by an infectious agent. Because there is a high incidence of human papillomavirus (HPV) infection, especially HPV-16, in homosexual men, we have sought HPV DNA sequences in Kaposi's sarcoma. We used the polymerase chain reaction with a primer pair specific for the highly conserved E6 region of HPV-16 to detect HPV-16 homologous DNA fragments in tumour tissues from 97 patients with KS and in KS-derived cell cultures. HPV DNA sequences were found in 11 of 69 KS skin tumours from homosexual men with AIDS-KS, in 3 of 11 KS biopsy specimens from homosexual men who had no clinical or laboratory evidence of HIV-infection, and in 5 of 17 KS skin lesions from HIV-1-negative elderly men and women with classic KS. The same primer pair amplified HPV-16 homologous fragments from two different continuous cell cultures derived from pleural effusion fluid of patients with pulmonary AIDS-KS and two continuous cell cultures derived from KS skin lesions. The findings suggest that HPV-16-related DNA sequences are associated with different forms of KS and may have a role in the pathogenesis of this neoplasm.