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Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata
Gene 188 (1997) 85–90
Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata Christine Herrmann, Herbert Zimmermann, Walter Volknandt * Biozentrum, J.W. Goethe-Universita¨t, Zoologisches Institut, AK Neurochemie, Marie-Curie-Str. 9/N210, D-60439 Frankfurt am Main, Germany Received 13 August 1996; revised 14 October 1996; accepted 14 October 1996
Abstract The major vault protein is the predominant constituent of vaults – ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved. Keywords: Recombinant DNA; Electric ray electric organ; Cholinergic nerve terminals; Vault ribonucleoprotein particle; mRNA size
1. Introduction Vaults are ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of yet unknown function (reviewed in Kickhoefer et al., 1996; Rome et al., 1991). The vault particle was originally identified as a barrel-shaped body in preparations of clathrincoated vesicles and named for its morphology reminiscent of the vaulted ceilings of cathedrals ( Kedersha and Rome, 1986). Vaults are multimeric protein complexes with a predominant member of about 100 kDa, the major vault protein, accounting for more than 70% of the total complex. Whereas the major vault protein is phylogenetically highly conserved between eukaryotes (Herrmann et al., 1996), the composition in the other and minor protein members of vaults differs ( Kedersha et al., 1990; Vasu et al., 1993). Recently, the human lung resistance-related protein (LRP) that has a high * Corresponding author. Fax +49 69 79829606; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DIG, digoxygenin; GAPDH, gene encoding glyceraldehyde-3-phosphate dehydrogenenase; kb, kilobase(s) or 1000 bp; LRP, lung resistance-related protein; MVP, major vault protein; MVP, gene encoding MVP; nt, nucleotide(s); ORF, open reading frame; RNP, ribonucleoprotein.
predictive value for resistance to chemotherapy in myeloid leukemia and ovarian carcinoma was found to be the human homologue of the major vault protein (Scheffer et al., 1995). One interesting feature of vaults is the presence of a single species of small RNA whose size varies between species ( Kickhoefer et al., 1993, 1996). By Northern blot analysis vault RNA was found in all mammalian tissues and cells analyzed with the lowest level of expression in brain tissue ( Kickhoefer et al., 1993). Vaults as described in rat and slime mold are similar in mass (12.9 MDa) and diameter (approx. 35–60 nm width and length; Chugani et al., 1993; Vasu and Rome, 1995; Herrmann et al., 1996). Recently we identified an electric ray homologue of the major vault protein, named MVP100 (Herrmann et al., 1996). In Torpedo marmorata synthesis of MVP100 is high in brain relative to liver. Highest levels are found in the electric lobe containing the cell bodies of the cholinergic neurons innervating the electric organ. MVP100 is localized abundantly in cholinergic nerve terminals of Torpedo in close proximity to synaptic vesicles. Thus, vaults are highly enriched in the electromotor system of electric rays where they are transported to the nerve terminal. In the present study we have further analyzed the cloned cDNA sequence of MVP100. The expression and
0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 7 81 - 0
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C. Herrmann et al./Gene 188 (1997) 85–90
genomic organization of MVP100 in electric ray is evaluated.
2. Materials and methods 2.1. cDNA library screening, cloning and sequencing For screening and isolating clones encoding MVP100 a lgt10 cDNA library constructed using mRNA derived from the electric lobe of the electric ray Discopyge ommata was used (gift from Dr. F. Rupp, Baltimore, MD, USA). 160 000 recombinants were screened using a 21-mer oligonucleotide as a probe designed from an aa sequence obtained by direct microsequencing of MVP100. The oligonucleotide was 3∞-tailed with DIG-dUTP kit (Boehringer-Mannheim, Mannheim, Germany). Two independent clones encoding MVP100 were sequenced from both ends. A clone of 1400 bp was used to isolate a 1800-bp and a full-length clone. Clones were digested by restriction mapping, subcloned and sequenced in both orientations. DNA sequencing was performed on double-stranded DNA using the dideoxy chain termination method (Pharmacia kit). Partial clones with more than 350 nucleotides were further sequenced using specific primers. Nucleotide and predicted amino-acid sequence were analyzed by computer searches of databases. 2.2. Northern blot analysis of MVP100 For Northern blot analysis mRNA was isolated from the electric lobe, electric organ and liver of Torpedo marmorata using an Oligotex Direct mRNA kit (Qiagen, Hilden, Germany). Equal amounts of mRNA as determined photometrically were separated on a 1% agarose formaldehyde gel, transferred onto hybond filters (Amersham, Braunschweig, Germany) and hybridized at 65°C in the presence of 5×SSC (0.75 M NaCl, 75 mM sodium citrate, pH 7.0) overnight, using various constructs of DIG-labelled RNA as a probe. Filters were washed twice at room temperature for 5 min with 2×SSC containing 0.1% SDS and twice at 65°C for 15 min with 0.2×SSC, 0.1% SDS. Hybridization signal was visualized by using anti-DIG alkaline phosphatase conjugated antibody and a chemiluminescent reagent (CSPD, both Boehringer-Mannheim) according to the manufacturer’s instructions. 2.3. Southern blot analysis of MVP100 Genomic DNA was isolated from Torpedo liver according to conventional methods as described by Strauss (1995). Equal amounts of genomic DNA as determined photometrically were separated on a 0.8% agarose gel, transferred onto hybond filters (Amersham)
and hybridized at 50°C in the presence of 5×SSC (0.75 M NaCl, 75 mM sodium citrate, pH 7.0) overnight. The probe for hybridization was DIG-labeled RNA of 2.7 kb in length containing the complete ORF. Filters were washed twice at room temperature for 5 min with 2×SSC containing 0.1% SDS and twice at 50°C for 15 min with 0.2×SSC, 0.1% SDS. Hybridization signal was visualized by using anti-DIG alkaline phosphatase-conjugated antibody (BoehringerMannheim) and nitro blue tetrazolium/5-bromo4-chloro-3-indolyl phosphate as a substrate according to the manufacturer’s instructions (Roth, Karlsruhe, Germany).
3. Results and discussion 3.1. Cloning and sequencing of the electric ray MVP100 The major vault protein (MVP100) was purified from electric ray electric organ and subjected to Edman degradation. An oligonucleotide (21-mer) was designed from the amino-acid sequence (DIETEAE ) obtained by direct microsequencing of MVP100 and used as a probe (see Fig. 1). By screening 160 000 recombinants two independent positive clones were obtained. A clone of 1400 bp was used to isolate a 1800-bp clone and a fulllength clone, named MVP100. The nt sequence and deduced aa sequence are illustrated in Fig. 1. The fulllength clone of 2717 nucleotides has a short 5∞-untranslated region without an in-frame stop codon, an ORF predicted to encode a protein of 852 aa, denoted MVP100, followed by a short 3∞-untranslated flanking region containing a polyadenylation signal and the poly(A) tail ( Fig. 1). The nucleotide sequence is rich in GC-nucleotides (61%). Evaluation of amino acids contained in the protein revealed a high contribution of the small aliphatic and hydrophobic residues alanine, valine and leucine (about 10% each) favoring multiple a-helical domains according to secondary structure predictions, whereas the sulfuric amino-acid residue cysteine, capable of constructing intramolecular disulfide bonds, contributes less than 1% to the amino-acid composition. The charged residues glutamic acid and arginine contribute to about 10 and 9%, respectively. The same percentage contribution in the above-mentioned amino acids is also found in the homologues of MVP100 in other species. The predicted MVP100 protein has a calculated molecular mass of 95.8 kDa and a pI of 5.5. 3.2. Northern blot analysis For Northern blot analysis mRNA was isolated from the electric lobe, electric organ and liver of Torpedo marmorata using an Oligotex Direct mRNA kit
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Fig. 1. Nucleotide sequence of the electric ray Discopyge ommata electric lobe MVP100 cDNA and deduced amino-acid sequence. ORF is in capital letters. Nucleotides and amino acids are numbered on the right. The end of the aa sequence is indicated by an asterisk. The oligonucleotide designed from the aa sequence (DIETEAE ) obtained by direct microsequencing of the protein as a probe for screening the cDNA library is underlined and the polyadenylation signal is indicated in bold. Clones were digested by restriction mapping, subcloned into pBluescript SKII and sequenced in both orientations. DNA sequencing was performed on double-stranded DNA using the dideoxy chain termination method. Partial clones with more than 350 nucleotides were further sequenced using specific primers. The sequence has been submitted to GenBank (accession No. X87771).
(Qiagen). Equal amounts of mRNA were loaded and hybridized using various constructs. In the electric lobe and liver one transcript of about 2.8 kb is labeled using a full-length DIG-labeled RNA as a probe (Fig. 2). The transcript size corresponds to the size of the cloned fulllength cDNA (cf. Fig. 1). The hybridization signal of MVP100 is stronger in the electric lobe as compared to liver, indicative of a higher expression in the neural tissue. This is in agreement with our earlier observation based on Western blotting that substantially more MVP100 protein is synthesized in the electric lobe than in liver (Herrmann et al., 1996). For comparison hybridization of the GAPDH transcript (about 1.6 kb) with a full-length DIG-labeled RNA probe was performed on the same filter (Fig. 2, lower panel ). When analyzing mRNA derived from Torpedo electric organ, a second strong hybridization signal at about 2 kb becomes apparent (Fig. 3, lanes 3 and 5). This occurs when either a full-length RNA or a RNA com-
prising 820 nt of the 3∞-end is used as a probe. In contrast, only the 2.8-kb transcript is labeled when RNA is used that comprises 724 nt of the 5∞-end (Fig. 3, lanes 1 and 2). For mRNA derived from the electric lobe only a single signal is obtained in all cases (Fig. 3, lanes 2, 4 and 6). The hybridization signal for GAPDH is shown for comparison ( Fig. 3, lanes 7 and 8). Non-specific hybridization to an unrelated transcript seems unlikely, because the lower hybridization signal is even stronger than that of the larger transcript. The strength of the signal indicates that it is a smaller and further processed transcript of MVP100, possibly a splice variant of MVP100 lacking about 800 nt of the 5∞-end. This transcript is probably not translated. Various specific antibodies against MVP100 detect only the 100-kDa protein in total tissue homogenates of the electric organ (Herrmann et al., 1996). It is noteworthy that for the isolation of mRNA from the electric organ considerably more tissue is needed than for brain. mRNA expression in this tissue is extremely low. Total
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Fig. 2. Northern blot analysis of Torpedo marmorata electric lobe (1) and liver (2) mRNA (0.9 mg). mRNAs were separated on a 1% agarose gel, blotted onto hybond filter (Amersham) and hybridized with a fulllength MVP100 DIG-labeled RNA probe. The molecular markers (RNA Markers, Promega) are given on the left in kb. The hybridization signal for GAPDH (at 1.6 kb) is marked with an arrow.
mRNA contents in the electric organ are about a 50-fold lower than in the electric lobe (w/w wet weight). The source of the two transcripts in the electric organ remains an open question, since major vault proteins are broadly distributed and produced in almost all eukaryotic cells (Izquierdo et al., 1996). In the electric organ, most of MVP100 is contained in the nerve terminals innervating the electrocytes. The respective mRNA is localized in the cell bodies of the electric lobes in the brain, where the protein is synthesized. The vault particle appears to be transported to the peripheral nerve endings (Herrmann et al., 1996). The source of the MVP100 transcript isolated from the electric organ originates presumably from endogenous non-neuronal cells. The electric organ is mainly composed of electrocytes. We
Fig. 3. Northern blot analysis of Torpedo marmorata electric organ ( lanes 1, 3, 5 and 7) and electric lobe ( lanes 2, 4, 6 and 8) mRNA (0.5 mg). mRNAs were separated on a 1% agarose gel, blotted onto hybond filter and hybridized with either full-length MVP100 ( lanes 3 and 4), 820 nt of the 3∞-end ( lanes 5 and 6), or 724 nt of the 5∞-end ( lanes 1 and 2) DIG-labeled RNA probes. For comparison the hybridization signal for GAPDH (at 1.6 kb) is shown ( lane 7, electric organ; lane 8, electric lobe). The size of the transcripts are given on the left (kb).
Fig. 4. Southern blot analysis of genomic Torpedo liver DNA (10 mg) digested with EcoRI ( lane 1), BamHI ( lane 2), HindIII ( lane 3) and SacI ( lane 4). Digested DNAs were separated on a 0.8% agarose gel, the DNA was blotted onto hybond filter and hybridized with a DIGlabeled full-length MVP100 RNA probe. The molecular markers are given on the left (kb).
demonstrated recently by immunohistochemistry that MVP100 is also localized in the proximity of the dorsal membrane of electrocytes ( Volknandt and Herrmann, 1997). Electron microscopical analysis with colloidalgold immunostaining confirmed this finding (unpublished observations). Moreover, immunostaining for MVP100 was also observed in tissue surrounding the axons, especially the perineurium. Thus, the isolated mRNA is presumably derived from a mixture of the various cell types present in the electric organ. 3.3. Southern blot analysis of MVP100 Genomic DNA was isolated from Torpedo liver according to conventional methods. The probe for hybridization was DIG-labeled RNA of 2.7 kb in length containing the complete ORF. By analyzing the genomic DNA using various restriction enzymes the size of the gene can be estimated to about 15–18 kb containing at least two introns. Restriction analysis with EcoRI yielded three fragments of approximately 9, 7 and 5 kb in size ( Fig. 4, lane 1). Since there are no internal EcoRI restriction sites in the entire mature RNA, these have to be located in introns. One BamHI (Fig. 4, lane 2) and one HindIII (Fig. 4, lane 3) restriction site are contained in the 3∞-coding region of the MVP100 mRNA. Restriction analysis with either BamHI or HindIII yielded three bands indicating at least one putative restriction site for these enzymes in one of the two introns. There is no restriction site for SacI in the MVP100 coding region. Digestion with SacI yielded one single band only ( Fig. 4, lane 4). These results are indicative of a single-copy gene. Similarly, the MVP104 gene from rat and the MvpA from the slime mold Dictyostelium discoideum have been reported to be single-copy genes ( Kickhoefer and Rome, 1994; Vasu
C. Herrmann et al./Gene 188 (1997) 85–90
et al., 1993). Whereas in higher eukaryotes only one isoform of the major vault protein has been reported, the slime mold appears to contain at least three different isoforms of MVPs ( Vasu and Rome, 1995). 3.4. MVP100 is the electric ray homolog of evolutionarily conserved major vault proteins By computer-based search in nucleotide and protein data bases, a high homology of the electric ray protein with the major vault protein of human, rat and slime mold was revealed. The evolutionary conservation of both the gene and the protein is illustrated in Fig. 5. Similarities of the cDNAs evaluated by alignment of the total number of bases comprising the coding region are given in percent of nt identity ( Fig. 5(A), top right). Nucleotide identity varies between 54 and 86%. At the protein level the aa identity is between 48 and 90% (Fig. 5(A), bottom left). The electric ray protein shows 69.1% aa identity (851 aa overlap) with the MVP proteins of human (Scheffer et al., 1995) and 68.7% (847 aa) of rat origin ( Kickhoefer and Rome, 1994), respectively. Furthermore, there is 55.8% aa identity (840 aa) and 52.1% (849 aa) with the two isoforms MvpA ( Vasu et al., 1993) and MvpB ( Vasu and Rome, 1995) of the slime mold Dictyostelium discoideum,
Fig. 5. Nucleotide and amino-acid comparison of electric ray MVP100 (MVP100) with the major vault proteins of rat, human and the two isoforms of Dictyostelium discoideum (D.d.). (A) The percentage of nt identity of the coding region of cDNAs is given in the right and upper part of the table, and that for aa identities in the lower and left part of the table. (B) Dendrogram of MVP clustering relationships. Percentage of nt identities of MVPs from electric ray with mammals (human and rat) or with slime mold (MvpA and B), and between the two mammalian or the two amoeba cDNAs are given on the axis.
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respectively. The identity between the mammalian proteins is 89.9% (868 aa) and between the amoeba isoforms is 60.4% (839 aa). The gene as well as the protein of vertebrates revealed similar identities to both of the two isoforms of the slime mold identified so far. Analysis of similarity is illustrated as a dendrogram of MVP clustering relationships in Fig. 5B. Recently, we showed by manual alignment of the predicted amino-acid sequences of all characterized MVPs that four long domains are phylogenetically highly conserved in the primary structure of the proteins ( Herrmann et al., 1996). Whereas the N-terminal parts of the proteins are conserved the C-termini show a hypervariable region interrupted by one small highly conserved domain. Secondary structure analysis predicts a very long a-helical domain in the architecture of the proteins located near the C-terminus, indicating an elongated tail in all proteins. MVPs contain numerous evolutionarily conserved putative phosphorylation sites for a variety of protein kinases, such as motifs for protein kinase C (4), casein kinase II (3), and tyrosine protein kinase (1).
4. Conclusions The major vault protein is the predominant constituent of a large cytoplasmic ribonucleoprotein (RNP) particle of yet undetermined function. The considerable abundance and high conservation argue for an important general function. The major vault proteins are phylogenetically conserved both with respect to the gene and to the primary structure of the protein. MVP100 has a few conserved myristoylation sites but none of the potential glycosylation sites is conserved between species. However, all major vault proteins contain numerous evolutionarily conserved consensus motifs for phosphorylation by several protein kinases. We showed recently that MVP100 becomes highly phosphorylated in vitro and in vivo ( Herrmann et al., 1996). This implies that phosphorylation is of relevance for controlling vault function in vivo. By immunofluorescence, evidence for a partial association of vaults with the nuclear pore complex has been derived (Chugani et al., 1993). Disruption of two of the three major vault proteins in Dictyostelium reveals only a mild growth defect under conditions of nutritional stress ( Vasu and Rome, 1995). Very recently a new avenue regarding the function of the major vault protein has been opened. The lung resistance-related protein (LRP) was found to be the human homologue of the major vault proteins of rat and slime mold (Scheffer et al., 1995). Our study further reveals that LRP is highly homologous also to the Torpedo MVP100. LRP overexpression was found to correlate with a poor response to chemotherapy in acute
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myeloid leukemia and ovarian carcinoma. It has been suggested that LRP, whose gene is closely located to the gene coding for the multidrug resistance-associated protein, may mediate drug resistance (Scheffer et al., 1995; Slovak et al., 1995). These observations suggest that major vault protein containing particles play a central role in cell homeostasis. The high contents of the MVP100 containing protein particles inside axon terminals implies new and yet unrecognized functional properties of this highly specialized compartment of the nerve cell.
Acknowledgement We thank Andrea Winter for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB169/A10).
References Chugani, D.C., Rome, L.H. and Kedersha, N.L. (1993) Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex. J. Cell Sci. 106, 23–29. Herrmann, C., Volknandt, W., Wittich, B., Kellner, R. and Zimmermann, H. (1996) The major vault protein (MVP100) is contained in cholinergic nerve terminals of electric ray electric organ. J. Biol. Chem. 271, 13908–13915. Izquierdo, M.A., Scheffer, G.L., Flens, M.J., Giacone, G., Broxtermann, H.J., Meijer, C.J.L.M., van der Falk, P. and Scheper, R.J. (1996) Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors. Am. J. Pathol. 148, 877–887.
Kedersha, N.L. and Rome, L.H. (1986) Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J. Cell Biol. 103, 699–709. Kedersha, N.L., Miquel, M.-C., Bittner, D. and Rome, L.H. (1990) Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J. Cell. Biol. 110, 895–901. Kickhoefer, V.A. and Rome, L.H. (1994) The sequence of a cDNA encoding the major vault protein from Rattus norvegicus. Gene 151, 257–260. Kickhoefer, V.A., Searles, R.P., Kedersha, N.L., Garber, M.E., Johnson, D.L. and Rome, L.H. (1993) Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III. J. Biol. Chem. 268, 7868–7873. Kickhoefer, V.A., Vasu, S.K. and Rome, L.H. (1996) Vaults are the answer, what is the question? Trends Cell Biol. 6, 174–178. Rome, L., Kedersha, N. and Chugani, D. (1991) Unlocking vaults: organelles in search of a function. Trends Cell Biol. 1, 47–50. Scheffer, G.L., Wijngaard, P.L.J., Flens, M.J., Izquierdo, M.A., Slovak, M.L., Pinedo, H.M., Meijer, C.J.L.M., Clevers, H.C. and Scheper, R.J. (1995) The drug resistance-related protein LRP is the human major vault protein. Nature Med. 1, 578–582. Slovak, M.L., Ho, J.P., Cole, S.P.C., Deeley, R.G., Greenberger, L., Devries, E.G.E., Broxterman, H.J., Scheffer, G.L. and Scheper, R.J. (1995) The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification. Cancer Res. 55, 4214–4219. Strauss, W.M. (1995) Preparation of genomic DNA from mammalian tissue. In: Ausubel, F.M., Brent, R. et al. ( Eds.), Current Protocols in Molecular Biology. Wiley, New York, NY. Vasu, S.K., Kedersha, N.L. and Rome, L.H. (1993) cDNA cloning and disruption of the major vault protein a gene (mvpA) in Dictyostelium discoideum. J. Biol. Chem. 268, 15356–15360. Vasu, S.K. and Rome, L.H. (1995) Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein. J. Biol. Chem. 270, 16588–16594. Volknandt, W. and Herrmann, C. (1997) The major protein of a large ribonucleoprotein particle ( VAULT ) is localized in nerve terminals. In: Teelken, W. and Korf, J. ( Eds.), Neurochemistry. Plenum, London, in press.
Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata Christine Herrmann, Herbert Zimmermann, Walter Volknandt * Biozentrum, J.W. Goethe-Universita¨t, Zoologisches Institut, AK Neurochemie, Marie-Curie-Str. 9/N210, D-60439 Frankfurt am Main, Germany Received 13 August 1996; revised 14 October 1996; accepted 14 October 1996
Abstract The major vault protein is the predominant constituent of vaults – ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved. Keywords: Recombinant DNA; Electric ray electric organ; Cholinergic nerve terminals; Vault ribonucleoprotein particle; mRNA size
1. Introduction Vaults are ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of yet unknown function (reviewed in Kickhoefer et al., 1996; Rome et al., 1991). The vault particle was originally identified as a barrel-shaped body in preparations of clathrincoated vesicles and named for its morphology reminiscent of the vaulted ceilings of cathedrals ( Kedersha and Rome, 1986). Vaults are multimeric protein complexes with a predominant member of about 100 kDa, the major vault protein, accounting for more than 70% of the total complex. Whereas the major vault protein is phylogenetically highly conserved between eukaryotes (Herrmann et al., 1996), the composition in the other and minor protein members of vaults differs ( Kedersha et al., 1990; Vasu et al., 1993). Recently, the human lung resistance-related protein (LRP) that has a high * Corresponding author. Fax +49 69 79829606; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DIG, digoxygenin; GAPDH, gene encoding glyceraldehyde-3-phosphate dehydrogenenase; kb, kilobase(s) or 1000 bp; LRP, lung resistance-related protein; MVP, major vault protein; MVP, gene encoding MVP; nt, nucleotide(s); ORF, open reading frame; RNP, ribonucleoprotein.
predictive value for resistance to chemotherapy in myeloid leukemia and ovarian carcinoma was found to be the human homologue of the major vault protein (Scheffer et al., 1995). One interesting feature of vaults is the presence of a single species of small RNA whose size varies between species ( Kickhoefer et al., 1993, 1996). By Northern blot analysis vault RNA was found in all mammalian tissues and cells analyzed with the lowest level of expression in brain tissue ( Kickhoefer et al., 1993). Vaults as described in rat and slime mold are similar in mass (12.9 MDa) and diameter (approx. 35–60 nm width and length; Chugani et al., 1993; Vasu and Rome, 1995; Herrmann et al., 1996). Recently we identified an electric ray homologue of the major vault protein, named MVP100 (Herrmann et al., 1996). In Torpedo marmorata synthesis of MVP100 is high in brain relative to liver. Highest levels are found in the electric lobe containing the cell bodies of the cholinergic neurons innervating the electric organ. MVP100 is localized abundantly in cholinergic nerve terminals of Torpedo in close proximity to synaptic vesicles. Thus, vaults are highly enriched in the electromotor system of electric rays where they are transported to the nerve terminal. In the present study we have further analyzed the cloned cDNA sequence of MVP100. The expression and
0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 7 81 - 0
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C. Herrmann et al./Gene 188 (1997) 85–90
genomic organization of MVP100 in electric ray is evaluated.
2. Materials and methods 2.1. cDNA library screening, cloning and sequencing For screening and isolating clones encoding MVP100 a lgt10 cDNA library constructed using mRNA derived from the electric lobe of the electric ray Discopyge ommata was used (gift from Dr. F. Rupp, Baltimore, MD, USA). 160 000 recombinants were screened using a 21-mer oligonucleotide as a probe designed from an aa sequence obtained by direct microsequencing of MVP100. The oligonucleotide was 3∞-tailed with DIG-dUTP kit (Boehringer-Mannheim, Mannheim, Germany). Two independent clones encoding MVP100 were sequenced from both ends. A clone of 1400 bp was used to isolate a 1800-bp and a full-length clone. Clones were digested by restriction mapping, subcloned and sequenced in both orientations. DNA sequencing was performed on double-stranded DNA using the dideoxy chain termination method (Pharmacia kit). Partial clones with more than 350 nucleotides were further sequenced using specific primers. Nucleotide and predicted amino-acid sequence were analyzed by computer searches of databases. 2.2. Northern blot analysis of MVP100 For Northern blot analysis mRNA was isolated from the electric lobe, electric organ and liver of Torpedo marmorata using an Oligotex Direct mRNA kit (Qiagen, Hilden, Germany). Equal amounts of mRNA as determined photometrically were separated on a 1% agarose formaldehyde gel, transferred onto hybond filters (Amersham, Braunschweig, Germany) and hybridized at 65°C in the presence of 5×SSC (0.75 M NaCl, 75 mM sodium citrate, pH 7.0) overnight, using various constructs of DIG-labelled RNA as a probe. Filters were washed twice at room temperature for 5 min with 2×SSC containing 0.1% SDS and twice at 65°C for 15 min with 0.2×SSC, 0.1% SDS. Hybridization signal was visualized by using anti-DIG alkaline phosphatase conjugated antibody and a chemiluminescent reagent (CSPD, both Boehringer-Mannheim) according to the manufacturer’s instructions. 2.3. Southern blot analysis of MVP100 Genomic DNA was isolated from Torpedo liver according to conventional methods as described by Strauss (1995). Equal amounts of genomic DNA as determined photometrically were separated on a 0.8% agarose gel, transferred onto hybond filters (Amersham)
and hybridized at 50°C in the presence of 5×SSC (0.75 M NaCl, 75 mM sodium citrate, pH 7.0) overnight. The probe for hybridization was DIG-labeled RNA of 2.7 kb in length containing the complete ORF. Filters were washed twice at room temperature for 5 min with 2×SSC containing 0.1% SDS and twice at 50°C for 15 min with 0.2×SSC, 0.1% SDS. Hybridization signal was visualized by using anti-DIG alkaline phosphatase-conjugated antibody (BoehringerMannheim) and nitro blue tetrazolium/5-bromo4-chloro-3-indolyl phosphate as a substrate according to the manufacturer’s instructions (Roth, Karlsruhe, Germany).
3. Results and discussion 3.1. Cloning and sequencing of the electric ray MVP100 The major vault protein (MVP100) was purified from electric ray electric organ and subjected to Edman degradation. An oligonucleotide (21-mer) was designed from the amino-acid sequence (DIETEAE ) obtained by direct microsequencing of MVP100 and used as a probe (see Fig. 1). By screening 160 000 recombinants two independent positive clones were obtained. A clone of 1400 bp was used to isolate a 1800-bp clone and a fulllength clone, named MVP100. The nt sequence and deduced aa sequence are illustrated in Fig. 1. The fulllength clone of 2717 nucleotides has a short 5∞-untranslated region without an in-frame stop codon, an ORF predicted to encode a protein of 852 aa, denoted MVP100, followed by a short 3∞-untranslated flanking region containing a polyadenylation signal and the poly(A) tail ( Fig. 1). The nucleotide sequence is rich in GC-nucleotides (61%). Evaluation of amino acids contained in the protein revealed a high contribution of the small aliphatic and hydrophobic residues alanine, valine and leucine (about 10% each) favoring multiple a-helical domains according to secondary structure predictions, whereas the sulfuric amino-acid residue cysteine, capable of constructing intramolecular disulfide bonds, contributes less than 1% to the amino-acid composition. The charged residues glutamic acid and arginine contribute to about 10 and 9%, respectively. The same percentage contribution in the above-mentioned amino acids is also found in the homologues of MVP100 in other species. The predicted MVP100 protein has a calculated molecular mass of 95.8 kDa and a pI of 5.5. 3.2. Northern blot analysis For Northern blot analysis mRNA was isolated from the electric lobe, electric organ and liver of Torpedo marmorata using an Oligotex Direct mRNA kit
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Fig. 1. Nucleotide sequence of the electric ray Discopyge ommata electric lobe MVP100 cDNA and deduced amino-acid sequence. ORF is in capital letters. Nucleotides and amino acids are numbered on the right. The end of the aa sequence is indicated by an asterisk. The oligonucleotide designed from the aa sequence (DIETEAE ) obtained by direct microsequencing of the protein as a probe for screening the cDNA library is underlined and the polyadenylation signal is indicated in bold. Clones were digested by restriction mapping, subcloned into pBluescript SKII and sequenced in both orientations. DNA sequencing was performed on double-stranded DNA using the dideoxy chain termination method. Partial clones with more than 350 nucleotides were further sequenced using specific primers. The sequence has been submitted to GenBank (accession No. X87771).
(Qiagen). Equal amounts of mRNA were loaded and hybridized using various constructs. In the electric lobe and liver one transcript of about 2.8 kb is labeled using a full-length DIG-labeled RNA as a probe (Fig. 2). The transcript size corresponds to the size of the cloned fulllength cDNA (cf. Fig. 1). The hybridization signal of MVP100 is stronger in the electric lobe as compared to liver, indicative of a higher expression in the neural tissue. This is in agreement with our earlier observation based on Western blotting that substantially more MVP100 protein is synthesized in the electric lobe than in liver (Herrmann et al., 1996). For comparison hybridization of the GAPDH transcript (about 1.6 kb) with a full-length DIG-labeled RNA probe was performed on the same filter (Fig. 2, lower panel ). When analyzing mRNA derived from Torpedo electric organ, a second strong hybridization signal at about 2 kb becomes apparent (Fig. 3, lanes 3 and 5). This occurs when either a full-length RNA or a RNA com-
prising 820 nt of the 3∞-end is used as a probe. In contrast, only the 2.8-kb transcript is labeled when RNA is used that comprises 724 nt of the 5∞-end (Fig. 3, lanes 1 and 2). For mRNA derived from the electric lobe only a single signal is obtained in all cases (Fig. 3, lanes 2, 4 and 6). The hybridization signal for GAPDH is shown for comparison ( Fig. 3, lanes 7 and 8). Non-specific hybridization to an unrelated transcript seems unlikely, because the lower hybridization signal is even stronger than that of the larger transcript. The strength of the signal indicates that it is a smaller and further processed transcript of MVP100, possibly a splice variant of MVP100 lacking about 800 nt of the 5∞-end. This transcript is probably not translated. Various specific antibodies against MVP100 detect only the 100-kDa protein in total tissue homogenates of the electric organ (Herrmann et al., 1996). It is noteworthy that for the isolation of mRNA from the electric organ considerably more tissue is needed than for brain. mRNA expression in this tissue is extremely low. Total
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Fig. 2. Northern blot analysis of Torpedo marmorata electric lobe (1) and liver (2) mRNA (0.9 mg). mRNAs were separated on a 1% agarose gel, blotted onto hybond filter (Amersham) and hybridized with a fulllength MVP100 DIG-labeled RNA probe. The molecular markers (RNA Markers, Promega) are given on the left in kb. The hybridization signal for GAPDH (at 1.6 kb) is marked with an arrow.
mRNA contents in the electric organ are about a 50-fold lower than in the electric lobe (w/w wet weight). The source of the two transcripts in the electric organ remains an open question, since major vault proteins are broadly distributed and produced in almost all eukaryotic cells (Izquierdo et al., 1996). In the electric organ, most of MVP100 is contained in the nerve terminals innervating the electrocytes. The respective mRNA is localized in the cell bodies of the electric lobes in the brain, where the protein is synthesized. The vault particle appears to be transported to the peripheral nerve endings (Herrmann et al., 1996). The source of the MVP100 transcript isolated from the electric organ originates presumably from endogenous non-neuronal cells. The electric organ is mainly composed of electrocytes. We
Fig. 3. Northern blot analysis of Torpedo marmorata electric organ ( lanes 1, 3, 5 and 7) and electric lobe ( lanes 2, 4, 6 and 8) mRNA (0.5 mg). mRNAs were separated on a 1% agarose gel, blotted onto hybond filter and hybridized with either full-length MVP100 ( lanes 3 and 4), 820 nt of the 3∞-end ( lanes 5 and 6), or 724 nt of the 5∞-end ( lanes 1 and 2) DIG-labeled RNA probes. For comparison the hybridization signal for GAPDH (at 1.6 kb) is shown ( lane 7, electric organ; lane 8, electric lobe). The size of the transcripts are given on the left (kb).
Fig. 4. Southern blot analysis of genomic Torpedo liver DNA (10 mg) digested with EcoRI ( lane 1), BamHI ( lane 2), HindIII ( lane 3) and SacI ( lane 4). Digested DNAs were separated on a 0.8% agarose gel, the DNA was blotted onto hybond filter and hybridized with a DIGlabeled full-length MVP100 RNA probe. The molecular markers are given on the left (kb).
demonstrated recently by immunohistochemistry that MVP100 is also localized in the proximity of the dorsal membrane of electrocytes ( Volknandt and Herrmann, 1997). Electron microscopical analysis with colloidalgold immunostaining confirmed this finding (unpublished observations). Moreover, immunostaining for MVP100 was also observed in tissue surrounding the axons, especially the perineurium. Thus, the isolated mRNA is presumably derived from a mixture of the various cell types present in the electric organ. 3.3. Southern blot analysis of MVP100 Genomic DNA was isolated from Torpedo liver according to conventional methods. The probe for hybridization was DIG-labeled RNA of 2.7 kb in length containing the complete ORF. By analyzing the genomic DNA using various restriction enzymes the size of the gene can be estimated to about 15–18 kb containing at least two introns. Restriction analysis with EcoRI yielded three fragments of approximately 9, 7 and 5 kb in size ( Fig. 4, lane 1). Since there are no internal EcoRI restriction sites in the entire mature RNA, these have to be located in introns. One BamHI (Fig. 4, lane 2) and one HindIII (Fig. 4, lane 3) restriction site are contained in the 3∞-coding region of the MVP100 mRNA. Restriction analysis with either BamHI or HindIII yielded three bands indicating at least one putative restriction site for these enzymes in one of the two introns. There is no restriction site for SacI in the MVP100 coding region. Digestion with SacI yielded one single band only ( Fig. 4, lane 4). These results are indicative of a single-copy gene. Similarly, the MVP104 gene from rat and the MvpA from the slime mold Dictyostelium discoideum have been reported to be single-copy genes ( Kickhoefer and Rome, 1994; Vasu
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et al., 1993). Whereas in higher eukaryotes only one isoform of the major vault protein has been reported, the slime mold appears to contain at least three different isoforms of MVPs ( Vasu and Rome, 1995). 3.4. MVP100 is the electric ray homolog of evolutionarily conserved major vault proteins By computer-based search in nucleotide and protein data bases, a high homology of the electric ray protein with the major vault protein of human, rat and slime mold was revealed. The evolutionary conservation of both the gene and the protein is illustrated in Fig. 5. Similarities of the cDNAs evaluated by alignment of the total number of bases comprising the coding region are given in percent of nt identity ( Fig. 5(A), top right). Nucleotide identity varies between 54 and 86%. At the protein level the aa identity is between 48 and 90% (Fig. 5(A), bottom left). The electric ray protein shows 69.1% aa identity (851 aa overlap) with the MVP proteins of human (Scheffer et al., 1995) and 68.7% (847 aa) of rat origin ( Kickhoefer and Rome, 1994), respectively. Furthermore, there is 55.8% aa identity (840 aa) and 52.1% (849 aa) with the two isoforms MvpA ( Vasu et al., 1993) and MvpB ( Vasu and Rome, 1995) of the slime mold Dictyostelium discoideum,
Fig. 5. Nucleotide and amino-acid comparison of electric ray MVP100 (MVP100) with the major vault proteins of rat, human and the two isoforms of Dictyostelium discoideum (D.d.). (A) The percentage of nt identity of the coding region of cDNAs is given in the right and upper part of the table, and that for aa identities in the lower and left part of the table. (B) Dendrogram of MVP clustering relationships. Percentage of nt identities of MVPs from electric ray with mammals (human and rat) or with slime mold (MvpA and B), and between the two mammalian or the two amoeba cDNAs are given on the axis.
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respectively. The identity between the mammalian proteins is 89.9% (868 aa) and between the amoeba isoforms is 60.4% (839 aa). The gene as well as the protein of vertebrates revealed similar identities to both of the two isoforms of the slime mold identified so far. Analysis of similarity is illustrated as a dendrogram of MVP clustering relationships in Fig. 5B. Recently, we showed by manual alignment of the predicted amino-acid sequences of all characterized MVPs that four long domains are phylogenetically highly conserved in the primary structure of the proteins ( Herrmann et al., 1996). Whereas the N-terminal parts of the proteins are conserved the C-termini show a hypervariable region interrupted by one small highly conserved domain. Secondary structure analysis predicts a very long a-helical domain in the architecture of the proteins located near the C-terminus, indicating an elongated tail in all proteins. MVPs contain numerous evolutionarily conserved putative phosphorylation sites for a variety of protein kinases, such as motifs for protein kinase C (4), casein kinase II (3), and tyrosine protein kinase (1).
4. Conclusions The major vault protein is the predominant constituent of a large cytoplasmic ribonucleoprotein (RNP) particle of yet undetermined function. The considerable abundance and high conservation argue for an important general function. The major vault proteins are phylogenetically conserved both with respect to the gene and to the primary structure of the protein. MVP100 has a few conserved myristoylation sites but none of the potential glycosylation sites is conserved between species. However, all major vault proteins contain numerous evolutionarily conserved consensus motifs for phosphorylation by several protein kinases. We showed recently that MVP100 becomes highly phosphorylated in vitro and in vivo ( Herrmann et al., 1996). This implies that phosphorylation is of relevance for controlling vault function in vivo. By immunofluorescence, evidence for a partial association of vaults with the nuclear pore complex has been derived (Chugani et al., 1993). Disruption of two of the three major vault proteins in Dictyostelium reveals only a mild growth defect under conditions of nutritional stress ( Vasu and Rome, 1995). Very recently a new avenue regarding the function of the major vault protein has been opened. The lung resistance-related protein (LRP) was found to be the human homologue of the major vault proteins of rat and slime mold (Scheffer et al., 1995). Our study further reveals that LRP is highly homologous also to the Torpedo MVP100. LRP overexpression was found to correlate with a poor response to chemotherapy in acute
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myeloid leukemia and ovarian carcinoma. It has been suggested that LRP, whose gene is closely located to the gene coding for the multidrug resistance-associated protein, may mediate drug resistance (Scheffer et al., 1995; Slovak et al., 1995). These observations suggest that major vault protein containing particles play a central role in cell homeostasis. The high contents of the MVP100 containing protein particles inside axon terminals implies new and yet unrecognized functional properties of this highly specialized compartment of the nerve cell.
Acknowledgement We thank Andrea Winter for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB169/A10).
References Chugani, D.C., Rome, L.H. and Kedersha, N.L. (1993) Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex. J. Cell Sci. 106, 23–29. Herrmann, C., Volknandt, W., Wittich, B., Kellner, R. and Zimmermann, H. (1996) The major vault protein (MVP100) is contained in cholinergic nerve terminals of electric ray electric organ. J. Biol. Chem. 271, 13908–13915. Izquierdo, M.A., Scheffer, G.L., Flens, M.J., Giacone, G., Broxtermann, H.J., Meijer, C.J.L.M., van der Falk, P. and Scheper, R.J. (1996) Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors. Am. J. Pathol. 148, 877–887.
Kedersha, N.L. and Rome, L.H. (1986) Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J. Cell Biol. 103, 699–709. Kedersha, N.L., Miquel, M.-C., Bittner, D. and Rome, L.H. (1990) Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J. Cell. Biol. 110, 895–901. Kickhoefer, V.A. and Rome, L.H. (1994) The sequence of a cDNA encoding the major vault protein from Rattus norvegicus. Gene 151, 257–260. Kickhoefer, V.A., Searles, R.P., Kedersha, N.L., Garber, M.E., Johnson, D.L. and Rome, L.H. (1993) Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III. J. Biol. Chem. 268, 7868–7873. Kickhoefer, V.A., Vasu, S.K. and Rome, L.H. (1996) Vaults are the answer, what is the question? Trends Cell Biol. 6, 174–178. Rome, L., Kedersha, N. and Chugani, D. (1991) Unlocking vaults: organelles in search of a function. Trends Cell Biol. 1, 47–50. Scheffer, G.L., Wijngaard, P.L.J., Flens, M.J., Izquierdo, M.A., Slovak, M.L., Pinedo, H.M., Meijer, C.J.L.M., Clevers, H.C. and Scheper, R.J. (1995) The drug resistance-related protein LRP is the human major vault protein. Nature Med. 1, 578–582. Slovak, M.L., Ho, J.P., Cole, S.P.C., Deeley, R.G., Greenberger, L., Devries, E.G.E., Broxterman, H.J., Scheffer, G.L. and Scheper, R.J. (1995) The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification. Cancer Res. 55, 4214–4219. Strauss, W.M. (1995) Preparation of genomic DNA from mammalian tissue. In: Ausubel, F.M., Brent, R. et al. ( Eds.), Current Protocols in Molecular Biology. Wiley, New York, NY. Vasu, S.K., Kedersha, N.L. and Rome, L.H. (1993) cDNA cloning and disruption of the major vault protein a gene (mvpA) in Dictyostelium discoideum. J. Biol. Chem. 268, 15356–15360. Vasu, S.K. and Rome, L.H. (1995) Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein. J. Biol. Chem. 270, 16588–16594. Volknandt, W. and Herrmann, C. (1997) The major protein of a large ribonucleoprotein particle ( VAULT ) is localized in nerve terminals. In: Teelken, W. and Korf, J. ( Eds.), Neurochemistry. Plenum, London, in press.