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At least 19 recordsLinked to original sources
PubMed2005

Preliminary analysis of two and three dimensional crystals of vault ribonucleoprotein particles.

Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults.

Animals↗
PubMed1997

Characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport.

Vaults are large ribonucleoprotein particles that have been identified in a wide range of eukaryotic organisms. Although present in thousands of copies per cell, their function remains unknown. In this report, we identify the major vault protein in sea urchins as a 107-kDa polypeptide that copurifies with microtubules and ribosomes. Although initially identified in microtubule preparations, the sea urchin major vault protein is not predominantly microtubule-associated in vivo. Rather, the sea urchin major vault protein is present throughout the cytoplasm in eggs and embryos and in the nucleus in adult somatic cells. Within the nucleus, the sea urchin major vault protein is concentrated in the region of the nucleolus and to punctate regions of the nuclear envelope. In addition, the vault protein localizes to short linear strings juxtaposed to the exterior of the nucleus and extending outward into the cytoplasm. Based on their copurification and intracellular distribution, vaults may be involved in the nucleocytoplasmic transport of ribosomes and/or mRNA.

Animals↗
PubMed1991

Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry.

The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function.

Animals↗
PubMed1993

Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex.

Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.

Animals↗
PubMed1999

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

Animals↗
PubMed1993

Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III.

Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These particles contain a unique small RNA (vault RNA (vRNA)). We have determined the sequence of the RNA associated with vaults purified from both rat and bullfrog. The rat vRNA is 141 bases in length, whereas the bullfrog vRNA is present as two highly related species of 89 and 94 bases. Despite the differences in length the predicted secondary structures of the three vRNAs are clearly related. All of the vRNAs contain sequences related to the internal promoter elements necessary for transcription by RNA polymerase III. The gene for the rat vRNA was isolated and sequenced from a rat genomic library, and its transcription by RNA polymerase III was verified using an in vitro transcription assay. The rat vRNA gene was efficiently transcribed in vitro, producing a single transcript of about 140 bases. Unlike most RNA polymerase III genes, the rat vRNA is present as a single copy gene and has a distinct tissue-specific expression pattern.

Animals↗
PubMed2003

Vaults: a ribonucleoprotein particle involved in drug resistance?

Vaults are ribonucleoprotein particles found in the cytoplasm of eucaryotic cells. The 13 MDa particles are composed of multiple copies of three proteins: an M(r) 100 000 major vault protein (MVP) and two minor vault proteins of M(r) 193 000 (vault poly-(ADP-ribose) polymerase) and M(r) 240 000 (telomerase-associated protein 1), as well as small untranslated RNA molecules of approximately 100 bases. Although the existence of vaults was first reported in the mid-1980s no function has yet been attributed to this organelle. The notion that vaults might play a role in drug resistance was suggested by the molecular identification of the lung resistance-related (LRP) protein as the human MVP. MVP/LRP was found to be overexpressed in many chemoresistant cancer cell lines and primary tumor samples of different histogenetic origin. Several, but not all, clinico-pathological studies showed that MVP expression at diagnosis was an independent adverse prognostic factor for response to chemotherapy. The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was therefore postulated that vaults contributed to drug resistance by transporting drugs away from their intracellular targets and/or the sequestration of drugs. Here, we review the current knowledge on the vault complex and critically discuss the evidence that links vaults to drug resistance.

Animals↗
PubMed2003

Unimpaired dendritic cell functions in MVP/LRP knockout mice.

Dendritic cells (DCs) act as mobile sentinels of the immune system. By stimulating T lymphocytes, DCs are pivotal for the initiation of both T- and B-cell-mediated immune responses. Recently, ribonucleoprotein particles (vaults) were found to be involved in the development and/or function of human DCs. To further investigate the role of vaults in DCs, we examined the effects of disruption of the major vault protein (MVP/LRP) on the development and antigen-presenting capacity of DCs, using our MVP/LRP knockout mouse model. Mononuclear bone marrow cells were isolated from wild-type and knockout mice and stimulated to differentiate to DCs. Like human DCs, the wild-type murine DC cultures strongly expressed MVP/LRP. Nevertheless, the MVP/LRP-deficient DCs developed normally and showed similar expression levels of several DC surface markers. No differences were observed in in vitro studies on the antigen uptake and presenting capacities of the wild-type and MVP/LRP knockout DCs. Moreover, immunization of the MVP/LRP-deficient mice with several T-cell antigens led to responses similar to those observed in the wild-type mice, indicating that the in vivo DC migration and antigen-presentation capacities are intact. Moreover, no differences were observed in the induction of the T cell-dependent humoral responses and orally induced peripheral T-cell tolerance. In conclusion, vaults are not required for primary DC functions. Their abundance in DCs may, however, still reflect basic roles in myeloid cell proliferation and DC development.

Administration, Oral↗
PubMed2004

Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.

Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

Amino Acid Sequence↗
PubMed2005

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.

Animals↗
PubMed2001

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.

Animals↗
PubMed2005

Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

Amino Acid Sequence↗
PubMed1998

Major vault protein of electric ray is a phosphoprotein.

The major vault protein is the predominant member of a large cytosolic ribonucleoprotein particle, named vaults. Vaults are abundant in nerve terminals of the electric organ of Torpedo marmorata. Negative staining of isolated vaults reveals particle dimensions of 45x65 nm in size. Comparison of the major vault protein (MVP100) from the two electric ray species Torpedo marmorata and Discopyge ommata reveals few microheterogeneities in amino acid sequence. Potential phosphorylation sites for various protein kinases are highly conserved. Phosphorylation studies demonstrate that the major vault protein of Torpedo is a substrate of various protein kinases. MVP100 is phosphorylated by protein tyrosine kinase in vivo and protein kinase C and casein kinase II in vitro. Inhibitors and activators of protein kinases specifically modulate the phosphorylation of MVP100.

Amino Acid Sequence↗
PubMed2005

Nuclear localization of the major vault protein in U373 cells.

The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of vault particles inside the nucleus has been convincingly reported only for sea urchin cells. We have addressed the potential nuclear localization of MVP in mammalian cells by employing confocal laser microscopy and cryo-immunoelectron microscopy. As revealed by immunostaining and by analysis of cells transfected with a construct encoding MVP and green fluorescent protein, MVP is present in both the cytosol and in the nucleus. Cryo-electron microscopy of human astroglioma U373 cells reveals clusters of immunogold particles at nuclear pores and in the nucleoplasm suggesting that nuclear MVP is associated with particulate structures. Quantification of the fluorescence observed in the cytosol and in the nuclei reveals that about 5% of the MVP in U373 cells is localized inside the nucleus. Our results further support the notion that part of the cellular MVP can enter the nucleus.

Astrocytoma↗
PubMed2001

Major vault protein is a substrate of endogenous protein kinases in CHO and PC12 cells.

Major vault protein (MVP) is the predominant member of a large cytosolic ribonucleoprotein particle, termed vault. We have previously shown that MVP derived from electric ray electric organ becomes phosphorylated by protein kinase C in vitro and by tyrosine kinase in vivo. Here we show that MVP from two mammalian cell lines (CHO and PC12 cell) becomes highly phosphorylated by endogenous protein kinases in cell-free systems. The susceptibility to protein kinases differs substantially from those observed in MVP derived from electric organ. Phosphorylation of MVP depends on the presence of Mg2+ and can be inhibited by the chelating agent EDTA. Inhibitors of casein kinase II attenuate the phosphorylation of MVP. In contrast to CHO cells, addition of recombinant casein kinase II enhances the phosphorylation of MVP in PC12 cells. Endogenous kinase activity is of particulate nature and copurifies with vault particles. Immuno-affinity purified vaults containing recombinant tagged MVP expressed in CHO cells reveal no autophosphorylation, suggesting that protein kinase activity is not an intrinsic property of vaults. Our results suggest that cell-specific phosphorylation of MVP may play a critical role in vault function.

Animals↗
PubMed2001

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.

Carrier Proteins↗
PubMed1996

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗