A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

Studies on the transcription, translation, and structure of alpha-actinin in Dictyostelium discoideum.

A clone coding for the F-actin cross-linking protein alpha-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in lambda gt11 with monoclonal antibodies specific for Dictyostelium alpha-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the alpha-actinin gene by comparing its nucleotide sequence with the amino acid sequence of tryptic peptides from purified alpha-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the alpha-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest continuous transcription of the alpha-actinin gene throughout the development of D. discoideum, up- and down-regulation of the levels of alpha-actinin mRNA and protein with maximum levels at the onset of aggregation, and a high diversity of alpha-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of alpha-actinin resides only at the functional sites, whereas the helical portions of the alpha-actinin molecule allow a higher level of diversity throughout evolution.

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration.

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.

The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation.

The 120-kD gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are homodimers and have extended rod-like configurations that are respectively approximately 35 and 40 nm long. Here we report the complete cDNA sequence of the 120-kD gelation factor which codes for a protein of 857 amino acids. Its calculated molecular mass is 92.2 kD which is considerably smaller than suggested by its mobility in SDS-PAGE. Analysis of the sequence shows a region that is highly homologous to D. discoideum alpha-actinin, chicken fibroblast alpha-actinin, and human dystrophin. This conserved domain probably represents an actin binding site that is connected to the rod-forming part of the molecule via a highly charged stretch of amino acids. Whereas the sequence of alpha-actinin (Noegel, A., W. Witke, and M. Schleicher. 1987. FEBS [Fed. Eur. Biochem. Soc.] Lett. 221:391-396) suggests that the extended rod domain of the molecule is based on four spectrin-like repeats with high alpha-helix potential, the rod domain of the 120-kD gelation factor is constructed from six 100-residue repeats that have a high content of glycine and proline residues and which, in contrast to alpha-actinin, do not appear to have a high alpha-helical content. These repeats show a distinctive pattern of regions that have high beta-sheet potential alternating with short zones rich in residues with a high potential for turns. This observation suggests that each 100-residue motif has a cross-beta conformation with approximately nine sheets arranged perpendicular to the long axis of the molecule. In the high beta-potential zones every second residue is often hydrophobic. In a cross-beta structure, this pattern would result in one side of the domain having a surface rich in hydrophobic side chains which could account for the dimerization of the 120-kD gelation factor subunits.

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex.

Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Tricorn protease--the core of a modular proteolytic system.

Large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. A tricorn-shaped protease was isolated from the archaeon Thermoplasma and was shown to form a multisubunit proteolytic complex. The 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities.

A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions.

Complementary DNA and genomic clones were isolated and sequenced corresponding to rat and human synaptophysin (p38), a major integral membrane protein of synaptic vesicles. The deduced amino acid sequences indicate an evolutionarily highly conserved protein that spans the membrane four times. Both amino and carboxyl termini face the cytoplasm, with the latter containing ten copies of a tyrosine-rich pentapeptide repeat. The structure of synaptophysin suggests that the protein may function as a channel in the synaptic vesicle membrane, with the carboxyl terminus serving as a binding site for cellular factors.

Targeting of the master receptor MOM19 to mitochondria.

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP.

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate.

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus.

BACKGROUND: The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS: We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Extracellularly occurring histone H1 mediates the binding of thyroglobulin to the cell surface of mouse macrophages.

Thyroglobulin is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. Clearance of thyroglobulin from the circulation occurs within the liver via internalization of thyroglobulin by macrophages. Here we have analyzed the interaction of thyroglobulin with the cell surface of J774 macrophages with the aim to identify the possible thyroglobulin-binding sites on macrophages. Binding of thyroglobulin to J774 cells was saturated at approximately 100 nM thyroglobulin with a Kd of 50 nM, and it was competed by the ligand itself. Preincubation of J774 cells with thyroglobulin resulted in downregulation of thyroglobulin-binding sites, indicating internalization of thyroglobulin and its binding proteins. By affinity chromatography, two proteins from J774 cells were identified as thyroglobulin-binding proteins with an apparent molecular mass of approximately 33 kD. Unexpectedly, both proteins were identified as histone H1 by protein sequencing. The occurrence of histone H1 at the plasma membrane was further proven by biotinylation or immunolabeling of J774 cells. The in vitro interaction between histone H1 and thyroglobulin was analyzed by surface plasmon resonance that revealed a Kd at 46 nM. In situ, histone H1 was colocalized to FITC-Tg-containing endocytic compartments of Kupffer cells, i.e., liver macrophages. We conclude that histone H1 is detectable at the cell surface of macrophages where it serves as a thyroglobulin-binding protein and mediates thyroglobulin endocytosis.

Identification of two related markers for common acute lymphoblastic leukemia as heat shock proteins.

By direct analysis of the polypeptide constituents of leukemic cells, we have previously detected several polypeptides that are restricted in their expression to acute lymphoblastic leukemia (ALL). In this study, we provide evidence that two polypeptides designated L2 and L4 are structurally related and represent novel markers for common ALL. Partial amino acid sequence analysis did not uncover differences between L2 and L4. The sequences obtained correspond to a previously cloned human gene designated hsp 27 that is expressed, following heat shock treatment, in a variety of cells. 32Pi incorporation studies indicate that L4 is an unphosphorylated form and L2 is a phosphorylated form of hsp27. The two forms were inducible by heat shock in leukemic and nonleukemic lymphoid cells. Thus, in acute leukemia, the common ALL subtype is uniquely characterized by the constitutive expression of a polypeptide that represents a major cellular phosphoprotein.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.