Assembly of vault-like particles in insect cells expressing only the major vault protein.

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.

Up-regulation of vaults may be necessary but not sufficient for multidrug resistance.

Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein-mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype. This implies a requirement of an additional factor(s) for vault-mediated MDR.

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

RNA location and modeling of a WD40 repeat domain within the vault.

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

The Mr 193,000 vault protein is up-regulated in multidrug-resistant cancer cell lines.

Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.

Analysis of sulfatide from rat cerebellum and multiple sclerosis white matter by negative ion electrospray mass spectrometry.

The accumulation of sulfatide (sulfatogalactosyl cerebroside) and changes in the sulfatide species present have been examined in the cerebellum of day 6-32 aged rats and in multiple sclerosis (MS) tissue samples. Negative ion electrospray mass spectrometry with daughter and parent ion analyses were used to distinguish the fatty acyl character in the amide linkage of sulfatide; measurement was done by selected ion and multiple reaction monitoring of individually identified sulfatide molecules. Sulfatide accumulation in rat cerebellum shows that 18:0- and hydroxylated 18:0-sulfatide are the first sulfatide molecules detectable. Very long fatty acyl chain sulfatide molecules (>20:0) are present at day 7 and the ratio of non-hydroxylated compared to hydroxylated sulfatide rises as the amount of non-hydroxylated sulfatide increases. 24:1-sulfatide accumulates at a ratio of about 3:1 over 24:0-sulfatide during active myelination. Analyses of the sulfatide in human tissue have shown differences between MS plaque tissues, normal appearing adjacent white matter and control tissues. The findings show that total sulfatide is reduced by 60% in the plaque matter and decreased 25% in adjacent normal appearing white matter. There are significant increases (P=0.05) in the amount of hydroxylation of sulfatide, demonstrated by an increase in the percentage of hydroxylated h24:0-sulfatide (hydroxy-lignoceroyl sulfatide).

Vaults and telomerase share a common subunit, TEP1.

Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly.

Cloning of a cDNA encoding a sequence-specific single-stranded-DNA-binding protein from Rattus norvegicus.

In this paper, we report the isolation of a cDNA clone encoding a sequence-specific single-stranded-DNA-binding protein (SSDP) from rat (Rattus norvegicus). The full-length nucleotide sequence was determined and encodes a 361 amino acid protein with a predicted molecular mass of 37.7 kDa. This clone has approximately 80% homology to a previously isolated partial cDNA clone for SSDP from chicken (Gallus gallus). Northern blot analysis revealed two transcripts of 2.0 and 3.0 kb. The protein appears to be evolutionarily highly conserved with > 97% identity between chicken, rat, mouse and human. Chicken SSDP has been proposed to be involved in the transcriptional regulation of the alpha 2(I) collagen gene.

The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

Structure of the vault, a ubiquitous celular component.

BACKGROUND: The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS: Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS: Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.

Role of axonal components during myelination.

Myelination is a multistep ordered process whereby Schwann cells in the peripheral nervous system (PNS) and oligodendrocytes in the central nervous system (CNS), produce and extend membranous processes that envelop axons. Mechanisms that regulate this complex process are not well understood. Advances in deciphering the regulatory components of myelination have been carried out primarily in the PNS and although the mechanisms for triggering and directing myelination are not known, it is well established that myelination does not occur in the absence of axons or axon/neuron-derived factors. This appears to be true both in PNS and CNS. Progress in understanding CNS myelinogenesis has been relatively slow because of the unavailability of a suitable culture system, which, in turn, is partly due to complexity in the cellular organization of the CNS. Though the myelin composition differs between PNS and CNS, the regulation of myelination seems to parallel rather than differ between these two systems. This article reviews the regulatory role of axonal components during myelination. The first half consists of an overview of in vitro and in vivo studies carried out in the nervous system. The second half discusses the use of a cerebellar slice culture system and generation of anti-axolemma monoclonal antibodies to investigate the role of axonal membrane components that participate in myelination. It also describes the characterization of an axonal protein involved in myelination.

Vaults are up-regulated in multidrug-resistant cancer cell lines.

Vaults are 13-MDa ribonucleoprotein particles composed largely of a 104-kDa protein, termed major vault protein or MVP, and a small vault RNA, vRNA. While MVP levels have been found to increase up to 15-fold in non-P-glycoprotein multidrug-resistant cell lines, the levels of vault particles have not been investigated. As both the function of vault particles and the mechanism of drug resistance in non-P-glycoprotein cells are unknown, we decided to determine whether vault synthesis was coupled to MDR. By cloning the human gene for vRNA and careful quantitation of the MVP and vRNA levels in MDR cells, we find that vRNA is in considerable excess to MVP. Sedimentation measurements of vault particles in multidrug resistance cells have indeed revealed up to a 15-fold increase in vault synthesis, coupled with a comparable shift of associated vRNA, demonstrating that vault formation is limited by expression of MVP or the minor vault proteins. The observation that vault synthesis is linked directly to multidrug resistance supports a direct role for vaults in drug resistance.

Axonal proteins involved in myelination: characterization of a collagen-like protein.

An anti-axolemma monoclonal antibody, designated G21.3, has been isolated in order to understand molecular mechanisms involved in myelination. Both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures. On Western blots of axolemma preparations, the antibody recognized 140- and 120-kD proteins. The present study involves the isolation and characterization of the G21.3 antigen. The G21.3-immunoreactive proteins of 140 and 120 kD were purified from the adult rat sciatic nerve and amino acid sequencing of these proteins revealed significant homology to alpha I and alpha II chains of collagen type I. Biochemical and Western blot analysis using pure collagen, collagen I antibody and collagenase D suggest that the antigen isolated from sciatic nerve is collagen. However, immunofluorescence studies using the G21.3 antibody, collagen I antibody, collagenase D and Northern blot analysis using a collagen probe do not fully support the view that the G21.3 antigen in the CNS is also a collagen. We conclude that the G21.3 antigen is a collagen-like protein involved in CNS myelination.

Vaults are the answer, what is the question?

Vaults are large cytoplasmic ribonucleoprotein (RNP) particles of eukaryotic cells, whose considerable abundance and striking evolutionary conservation argue for an important general cellular function. Early studies on vaults focused on the structural features and cellular distribution of the particle and will only be summarized briefly here. In this article, we discuss the molecular characterization of vault components and describe genetic studies carried out in Dictyostelium. The recent finding that the major vault protein is elevated in non-P-glycoprotein multidrug resistant cancer cells has direct implications concerning the function of the vault particle and indicates a potential role for vaults in resistance of tumour cells to anticancer drugs.

Relationship of LRP-human major vault protein to in vitro and clinical resistance to anticancer drugs.

Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cells lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor for in vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.

Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein.

Vaults are large cytoplasmic ribonucleoprotein particles that are highly conserved in both morphology and protein composition. Protein components of vaults isolated from Dictyostelium discoideum migrate on SDS-polyacrylamide gels as two bands, one at 94 kDa (MvpA) and the other at 92 kDa (MvpB). An MvpB cDNA clone was isolated from a Dictyostelium expression library. MvpB shares 60% identity with MvpA at the amino acid level. This cDNA has been used to disrupt the single mvpB gene in both wild-type and mvpA- genetic backgrounds. Although the mvp- mutant lines are viable, they show that loss of MvpA and/or MvpB interferes with vault function sufficiently to impede growth under conditions of nutritional stress. The resulting mutant cell lines reach stationary phase in suspension culture at one-third of the density of wild-type cells. Ovoid structures isolated from mvp- single mutant lines represent what remains of vaults in these cells. Similar ovoid structures isolated from the mvpA- mvpB- line copurify with a newly identified protein of 92 kDa (MvpC), which lacks cross-reactivity with currently available anti-vault antibodies. Our results indicate that the major vault proteins are necessary for optimal cell growth in Dictyostelium and reveal an unanticipated complexity in vault composition.

Cloning and sequence of the cDNA encoding the rat oligodendrocyte integrin beta 1 subunit.

We have isolated a cDNA clone encoding an integrin (Itg) beta-subunit polypeptide from a rat cerebral cortex oligodendroglia cDNA library. This beta-subunit was previously designated beta OL (OL for oligodendroglia), since it had an M(r) that differed from that of the rat fibroblast beta 1 protein. The complete nucleotide sequence encoding the signal sequence and mature protein was determined. Comparison of the deduced amino acid (aa) sequence of beta OL to those of integrin beta subunits from other species revealed that beta OL is a member of the integrin (Itg) beta 1 class. The aa sequence of beta OL shows 78.5-97.9% similarity to beta 1 chains from four other species and displays all the features characteristic of the Itg beta subunits, indicating the high degree of structural conservation seen in this subunit of the Itg receptors.

The sequence of a cDNA encoding the major vault protein from Rattus norvegicus.

We have isolated a cDNA clone encoding the 104-kDa major vault protein (MVP) from Rattus norvegicus. The complete nucleotide sequence was determined. Comparison of the deduced amino acid (aa) sequence to that of MvpA from Dictyostelium discoideum revealed that the proteins share about 57% aa identity. Southern blot analysis indicates that the rat MVP is a single-copy gene.

The rat vault RNA gene contains a unique RNA polymerase III promoter composed of both external and internal elements that function synergistically.

A novel gene transcribed by RNA polymerase (pol) III has been recently identified that produces an RNA component of a large cytoplasmic ribonucleoprotein complex (Kickhoefer, V. A., Searles, R. P., Kedersha, N. L., Garber, M. E., Johnson, D. L., and Rome, L. H. (1993) J. Biol. Chem. 268, 7868-7873). Since sequence analysis revealed that this gene contains promoter elements from two different classes of RNA pol III gene promoters, we examined the function of the 5'-flanking type-3 and internal type-2 sequences on transcription activity and the production of stable transcription complexes. We find that the vRNA gene contains a novel RNA pol III promoter, where both the external and internal sequences are essential for template activity and for the productive interaction of TFIIIC with the internal elements. Thus, the vRNA gene represents the first example of a template that requires both type-2 and type-3 promoter elements that appear to function synergistically in the formation of productive transcription complexes. We have further examined the function of the unique arrangement of an internal A box and two B box elements. We find that at least one B element is required for template activity. In the absence of the 5'-flanking sequence the presence of both B elements inhibits transcription and the binding of TFIIIC. The formation of active complexes is restored when either the B2 element is inactivated or the distance separating the two B elements is increased. Therefore, the B2 element appears to negatively regulate template activity in the absence of the upstream sequences. This unique RNA pol III promoter arrangement may provide a novel mechanism for the regulation of vRNA gene activity.