minor proteins of 210, 192, and 54 kDa and a major vault protein (MVP)' of 104 kDa, which accounts for 75% of the particle mass and is believed to make up the ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 21, Issue of July 25, pp. 15356-15360,1993 Printed in U.S. A.
cDNA Cloning and Disruption of the Major Vault Protein (I! Gene (mupA) in Dictyostelium discoideum” (Received for publication, December 16, 1992, and in revised form, March 4, 1993)
Sanjay K.Vasus, Nancy L. Kedershaa, and Leonard H. Romesll From the $.Department of Biological Chemistry, University of California School of Medicine, Los Angeles, California90024-1737 and SImmunoGen, Inc., Cambridge, Massachusetts 02139-4239
Vaults arelarge cytoplasmic ribonucleoprotein par- of vaults include macrophages and Dictyosteliurn amoeba (5). ticles found in nearly all eukaryotic cells. Dictyoste- Dictyostelium vaultsare morphologically similar to those lium vaults contain two major proteins, MVPa (94.2 found in other organisms. However, Dictyostelium is one of kDa) and MVPB (-92 kDa). Using an anti-rat vault few organisms in which the MVP is actually two proteins (3). antibody, we screened aDictyostelium cDNA expresThe discovery of vaults in Dictyostelium offered the opporsion library and isolated 2.8-kilobase a pair clone that tunity to conduct molecular genetic investigations into vault contained asingle full-length reading frame. The identity of the clone was established by the presenceof a structure and function by providing a cell type that allows gene disruption by homologous recombination (6-10). Since predicted 20-amino acidsequenceidenticaltothat the MVPs representthe bulk of the particle mass, eliminating found in a peptide sequenced from purified MVPa. We have disrupted thesingle copy gene using homologous these proteins should disrupt vault structure and function. recombination and have demonstratedloss a of MVPa. The availability of a vault mutant Dictyostelium cell line would provide a unique opportunity to study the physiological Althoughthe cells still produceMVPB, they donot contain characteristic vaultparticles, suggesting that significance of losing a structure that is normally abundant MVPa is required for normal vault structure. These in this organism and expressed throughout its development cells should be a valuable tool for elucidating the func(3). tion of vaults. In this study, we report the isolation of a cDNA clone
Vaults are cytoplasmic ribonucleoprotein particles of unknown function. They were originally isolated from rat liver and visualized by electron microscopy after negative staining (1). Their barrel-shaped morphology is conserved between species as phylogenetically diverse as mammals, amphibians, avians, andthe lower eukaryote Dictyosteliumdiscoideum (reviewed in Ref. 2). The protein composition of vaults is similarly conserved (3). Rat liver vaults arecomposed of three minor proteins of 210, 192, and 54 kDa and a major vault protein (MVP)’ of 104 kDa, which accounts for 75% of the particle mass and is believed to make up the petals or side panels of the vault (1).Rat vaults also include a small RNA molecule of 141 bases known as the vRNA (1). Two small vault-associated RNAs with structural similarity to the rat vRNA have recently been isolated from bullfrog liver, suggesting that the vRNA might be an important component of vaults (4). Although vaults have been found ina wide variety of eukaryotic cells, their abundance varies greatly between different tissues and cell types (1-5). The most abundant sources
* This work was supported by United States Public Health Service Grant GM38097. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted to the GenBankTM/EMBL DataBank withaccessionnumberfs) LO8646 7 To whom correspondence should be addressed: Dept. of Biological Chemistry, 33-257 CHS, UCLA School of Medicine, Los Angeles, CA 90024-1737.Tel.: 310-825-0709; Fax: 310-206-5272. The abbreviations used are: MVP, major vault protein; vRNA, vault RNA kb, kilobase pair(s); bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.
encoding MVPa. The gene for MVPa (mupA) has been mutated by homologous recombination to generate an MVPanegative cell line. We demonstrate interruption of the genomic copy of the mvpA gene and a failure of this mutant line to produce mvpA mRNA, the MVPa gene product, or characteristic vault particles. However, even in the absence of the MVPaprotein, thiscell line is able to express a particle that contains the MVPp protein. As this MVPa-negative cell line exhibits apparently normal growth and development, it seems likely that the potentially redundant nature of MVPa and MVPP allowsMVPP-containing vaults to carry out vault function even in the absence of MVPa protein. EXPERIMENTALPROCEDURES
Antibody Production-Vaults (100 pg) purified from Dictyostelium (5) were emulsified in Freund’s complete adjuvant (Sigma) and injected into a New Zealand White rabbit. Boosts were done monthly using 50pgof vaults in Freund‘s incomplete adjuvant (Sigma). Affinity purification of anti-vault antibodies from rabbit serum was performed on avault-Sepharose CL4B column as previously described (3). cDNA Cloning, Sequencing, and Clone Verification-A Dictyostelium cDNA expression library in X gtll (Clontech) was screened using an anti-ratvault polyclonal antibody. Due to thepresence of internal EcoRI sites, DNA containing the cDNA insert was isolated by digestion of the X clone with KpnIISstI. Restriction at these sites, which are only found within the X DNA about 1 kb on either side of the cDNA insert, released a fragment that could be easily gel-purified. X DNA was removed from the cDNA by digestion with B a n I I I H M leaving e 1 5 bp of X DNA. This latter digestion could not be carried out initially due to thepresence of multiple BanIIIHhaI sites throughout the X gtll vector. The resulting 2.8-kb fragment was blunted and subcloned into the SmaI site of the phagemid, pUC118 (111,generating pMVPa. Dictvostelium vaults were electrouhoresed on an 8% SDS-PAGE g e l ( l 5 and MVPa protein was elutkd. The protein was reapplied to a 12.5% SDS-PAGE gel in the presence of V8 protease. Peptides were electroblotted to polyvinylidene difluoride membrane sequenced by
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60-70% helical. The protein is predicted to have a pI of 6.7, an estimate in agreement with empirical measurements on thenative major vaultproteinfromrat (1). Searches of GenBank and EMBL data bases at both nucleic acid and amino acid levels didnot show theMVPa cDNA to be significantly similar to any known sequence. The predicted protein sequence was scanned for thepresence of known motifs using the program MOTIFS (14). Although potential phosphorylation and glycosylation sites were revealed, there isno evidence that MVPa is modified. The Dictyostelium cDNA fails to detectmup genes on Southern blotsof genomic DNA froma variety of species includingrat (data not shown). This lack of homology is likely to be due at least in part to the strongcodon usage bias of Dictyostelium. Physical Map of the mupA Locus-A map of restriction endonuclease recognition sites is shown (Fig. 2B). ThemvpA locus is contained within a 7-kb HindIII fragment (Fig. 2 A ) . Digestion with several endonucleases that do not cut within the cDNA yielded single bands of comparable intensity, suggesting that mvpA is a single copy gene (data not shown). Only onecopy of the coding sequenceis present in the HindIII fragment, asa BamHI/HindIII double digest dividesthe gene into a 4.3-kb and a 2.7-kb fragment, each of which is recognized by unique cDNA probes (Fig. 2 A ) . Disruption of the mvpA Locus and Isolation of MVPanegative Lines-We took advantage of the high frequency of homologous recombination in Dictyostelium to target a selectable marker to the mvpA genomic locus. The neomycin resistance cassette under Dictyostelium actin-15 promoter and terminator elements was inserted into the 5’ BglII site of pMVPa, disrupting the MVPacoding sequence and creating the plasmid pMVPaNEO (Fig. 3). This plasmid was transfected into the haploid strain, Ax-4, and G418-resistant colonies were selected onsoft agarose plates (16). Potential transformants were isolated and grown in liquid culture in the presence of G418 (10 mg/ml) to select for stable drugresistantlines.The survivorswerescreenedby Western analysis for the presence of MVPa. One line, which we designate M3, appeared to express no immunoreactive MVPa protein (Fig. 4). MVPp protein level in M3 cells is reduced to approximately 20% of the wild-type level as determined by densitometry on Western blots using newly our produced antiDictyostelium vaultantibody(see“ExperimentalProcedures’’), which detects MVPa and MVPPequally well. Physical Map of the Disrupted mupA- Gene in M3 CellsT o demonstrate correct targetingof the knock-out construct into the mvpA gene, we showed a loss of the 7-kb HindIII fragment that contains the wild-type gene and restrictionmapped the disruptedgene. Digests of mutant genomic DNA revealed a duplication of MVPa coding sequenceat themvpARESULTS locus (Fig. 5A). Digestion patterns were consistent with integration of the plasmid pMVPaNEO via homologous recomIsolation of a cDNA Clone Encoding mupA-A polyclonal bination between the 5’ cDNA sequence and the5’ end of the rabbit anti-rat vault antibody (1) was used to screen a Dictyostelium cDNAexpressionlibrary.Thisantibodyhas a gene. The plasmid disrupted the gene by inserting the drug resistance cassette and vector sequence into themiddle of the much greater affinity for MVPa than for MVPP. A 2.8-kb clone (shown in Fig. 1) was isolated, sequenced, and found to genomic coding sequence. An intact copy of the coding sequence was recreateddownstream of theintegration site. contain asingle openreadingframe, 2530 bpinlength, of a promoter or otherregulatory followed by a polyadenylation signal. The open reading frame However, due to the absence mvpA transcript encodes a 94.2-kDa polypeptide. Aminoacid sequence analysis sequences, it appears to be inactive as no can be detected on Northern blots of total M3 RNA (Fig. 6). derived from a V8 proteolytic fragment of gel-purified DicThe viability of this mvpA- cell line indicates that mvpA is tyostelium MVPa was found to match amino acids 423-444 not an essential gene in an mvpB genetic background. in the cDNA open reading frame, thereby establishing its (see Fig. identity as the largerof the two major vault proteins Characterization of MVPP-containing Structures from M3 1).Computer analysisof the sequence using the University of Cells”M3 cells were axenically cultured to a density of 5 X Wisconsin Genetics Computer Group software package (14) lo6 cells/ml and taken through the same cell fractionation reveals a generally hydrophilic polypeptide predicted to be scheme previously reported for purification of vaults from
Dr. Audree Fowlerat the UCLA Biological Chemistry Microsequencing Facility. DNA sequencing was done on single-stranded DNA (11)using the dideoxv chain termination method (13). The cDNA was sequenced twice both directions using a series of random subclones generated by multiple restriction enzyme digestions.DNA and predicted amino acid sequence were analyzed on both DNASTAR (DNASTAR, Inc., Madison, WI) and the University of Wisconsin Genetics Computer Group software package(14). DictyosteliumCulture andTransfections-Thehaploid strain of Dictyostelium, Ax-4, was a gift from Dr. DavidKnecht (Universityof Connecticut). Cell linemaintenance,culture, and preparation of genomic DNA and RNA were performedas described (7,15).Calcium phosphate-mediatedtransfections were performed according to published protocols (16). Western Analysis-Cells were grown axenically to a density of lo6 cells/ml. Cells (lo7) were pelleted and resuspended in SDS sample loading buffer. Twentypg of total protein was subject to SDS-PAGE on an 8%/10% step running gel with a 3% stack(12).Electrophoresed proteins were electroblotted onto nitrocellulose and blocked with 4% nonfat dry milk. Blots were incubated1 h with a 200-fold dilution of an affinity-purified anti-Dictyosteliurn vault polyclonal antibody followedby incubation with horseradishperoxidase-conjugatedgoat anti-rabbit affinity-purifiedantibody (Bio-Rad) anddevelopment with 4-chloro-1-naphthol andhydrogen peroxide. Southern and Northern Analysis-For Southern blots, 10pg of genomicDNAwasdigestedwith the appropriaterestriction enzyme(s),electrophoresed on a 1%agarose gel, and transferred overnight to nylon membrane (ICN Biotrans+) in 0.4 N NaOH (17-19). The blot was prehybridized overnight at 65 “C in 6 X SSPE, 5 X Denhardt’s solution,0.35% SDS, and 0.2 mg/ml salmon sperm DNA (20). Randomly primed probe (specific activity 2 X lo9 cpm/pg) was added at 2 X IO6 cpm/ml and the blot hybridized overnight at the same temperature.The filter was washed finally for1 h in 0.1 X SSC, 0.1% SDS at 65 “C and exposed to Kodak XAR-5 film at -80 “C. Northern blots included 10 pg of total RNA separated on a 1% agarose, 1%formaldehydegel and transferred to nylon membrane (ICN Biotrans+) in 10 X SSC (18). Hybridization and washing conditions were as described for Southern analysis except BSA hybridization buffer (1% BSA, 0.5 M sodium phosphate buffer pH 7.0, 1 mM EDTA, and 7% SDS) containing 200pg/ml yeast RNAwas used (21). Plasmid Constructs-All enzymes were purchased from Promega or Boehringer Mannheim. The cDNA clone pMVPa was generated as described above. The plasmid, pNEOMLS-, was a gift from Dr. James Spudich (Stanford University). The gene knock-outvector pMVPaNEO was constructed by digesting pMVPa with BglII followed by gel purification. The linearized construct was blunted with Klenow fragment and treated with calf intestine alkaline phosphatase. A cassette containingthe bacterial neomycin resistance coding sequence (aminoglycoside phosphotransferase (3’-11)) under the Dictyostelium actin-15 promoter (6) wasisolatedfrom the plasmid, pNEOMLS-, by ScaIIEcoRV digestion and ligated intothe BglII site (position 1467;seeFig. 1) in the digestedandbluntedpMVPa generating pMVPaNEO. Restriction mapping of the isolated plasmid revealed that the MVPa BglII fragment (positions 1467-1691) was not lostasoriginally thought and that the neomycin resistance cassette was inserted into pMVPa at the 5’ BglII site (position 1467).
Cloning Disruption and
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ofMajor Vault Protein the
ATG GCCGATTTA
AAC T C TG T TA T T
GTTTTTTAAATTATTATTTATAAATAATCTTTATCTTTAACTCTATATATAT~TA AGA ATT AAA CCA TTT CAT TTCATT CAC GTATTA GAT AAT AAT ACA AAT GTTACT
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AGA ATT GAA GAA GAA ATC AAA GCA ACC ATCATT
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GCA AGA AAA GCTGGT GAA GAG TGG TTAGTTCGT CAA CATGGTGCCTACCTCCCAGGTGTCGAT V A R K A G E E W L V R Q H G A Y L P G V D GAA ATTGTAAATGCCTACGTCTTAACT GAC AAG AAA GCA CTC CAC CTC AAA GCCACT AAA ACC T T T
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CAA ATC TATGATTAC
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AAC CCA R K G E L A F F L N P ACC GAA CAA GAA GCA CTCTTACTCCGTGCT AAA T E Q E A L L L R A K TGG ATG ATCTATGGT CCA TGTGATTATGTT CCA
ACC GTTCTCTCTTTATCTGGTGAT T V L S L S G ATG ACTGAT GTTGTCATTGTC
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GATGATTTC CAC AAA CGTTCCGCT GAA GTCATCCGT D D F H K R S A E V I R AAC TTCTCTTTCAATTCA AAC AAC CTTGTC ATCACC
CAA TCCGTTTTTGGTTTA GAT GAA TCTGGT GAA GTC CGT AAG Q S V F G L D E S G E V R K AAC ATTGATATT CAA TCTGTT GAA CCA GTCGAT CAA CGTACT
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CAA AAA TCTGTC
CGTGATTCACTC
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A
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GATTTATTA CAA CTC CAA GCT CAA D L L Q L Q A Q GAA GCTGCT AAC ATTGCTGCT GAA E A A N I A A E T C T GAA ATTTCTCTCTTA AAA GCC S E I S L L K A AAA CAA TCTGATCTCGCT GAA ATT
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ACCGACGGT
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AAA TCA CAA GAA GCTGCTGCTCGTCAT
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ACC GCT GAA T A E CAA GCC ACC Q A T AAA TCAATTGAT K S I D GAA TCCATTGGT
V
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GCT A GCT A
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CAA GGTTTGGGTCTT
GCC AAC GGTATCATTGGT
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GTC GAG TCAACTGGT CAA V E S T G Q AAA CAA GCT GAA TTA AAA K Q A E L K AAT GAA CTCTCATAC CAA N E L S Y Q AAA TTC AAA OCAATCGTT
AAT GAA ATG CAA GCT AAA TTATTA AAC CTTTTCGATACT
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ATT GAA ATCACCACC
TCAGCT GCA S A A GCCAAT GTT A N V AAG AGA GAA K R E GAA GCCTCC
AAA TCACCACTC
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AGA CTC GAA CGT CAA AAG ATT CAC GAT GAA GCT CAA GCA GAG TTAGCT
CGT AAA R K ACTGCT T A TCA GAA S E CTC ACC
CTC AAA TCAATTGCTTGTGCTGGT
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CAA TTGGCC
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GCT A AAG K GAA E AGA
CAA Q ATT I CTC L GAT
GCT A CGT R GAA E ACC
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AAA TCATTT
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ATG ATC
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AAT AAC AAT CAA ATG GTA AAC ATG
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TCAAATGCTGCC AAA CAA TCTGGT AGA AGA AATTAA AAAAAAATAATAATCACAAATAACATTTTCAAATACEGU S N A A K Q S G R R N AAAAAAAAAAGAATGAGATACATTCTTTTTTTTTATTTGATMTAAAATAIVlAAACTATTGTA
60 138 26 216 52 294 78 372 104 450 1 30 528 156 606 182 684 208 762 234 840 260 918 286 996 312 1074 338 1152 364 1230 390 1308 4 16 1386 442 1464 468 154 494 1620 520 1698 546 1776 572 1854 598 1932 624 2010 650 2088 676 2166 702 2244 728 2322 754 2400 780 2478 806 2556 832 2647 843 2750 2783
FIG. 1. Nucleotide and deduced amino acid sequences of the MVPa cDNA. The amino acids confirmed by peptide sequence data are underlined. The 5’ BgZII site into which the neomycin resistance cassette was inserted is double underlined, and the polyadenylation site is shown in boldface.
wild-type Dictyostelium (3, 5 ) . In four separate purification attempts, we could find no characteristic vault particles. However, we were able to identify a structure in these cells that co-fractionates withMVPp protein. Although present in con-
siderably reduced quantity relative to vault levels in normal cells, these structures could be visualized by electron microscopy after negative staining. These particles (Fig. 7 B ) lack the lobular morphology of normal vaults (Fig. 7 A ) .The ovoid
Cloning and
Disruption of the Major Vault Protein
15359
A .
108 -
BmHl 6 -
4-
x
B.
I+
Lane 1:
Lane 2: Lane 3 Lane 4 Lane5
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H
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X B
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7.0
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,
1 -2
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3.
200
I
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FIG.3. mvpA gene disruption vector. The bacterial neomycin resistance coding sequence (black region) fused to the Dictyosteliurn actin-15 promoter (24) was inserted into the 5’ BgnI site of the MVPa cDNA (gray region). Arrows indicate the translation start sites of the MVPa and NEO’ coding sequences. This vector was introduced into wild-type Ax-4 cells via calcium phosphate transfection and integrated into the genome at themupA locus. Parentheses around the BglII sites indicate that they were destroyed by the blunting reaction and insertion of the NEO’ coding sequence.
10.0
85
8.5
I
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MVPa ‘MVm
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FIG.2. A , Southern blot of Dictyostelium genomic DNA. Wildtype genomic DNA (10pg) was digested with BamHI (lane1 ), HindIII (lane2 ) , BamHI/HindIII (lane3), and BamHIIXhoI (lanes4 and 5). Probe A is a 900-bp BamHI fragment specific to the 5’ end of the cDNA. Probe B is an 200-bp BglII cDNA fragment from the 3’ half of the cDNA (see Fig. 2B). Lunes 1-4 were hybridized with probe B. Lane 5 was hybridized with probes A and B. B, restriction map of the wild-type mvpA locus. Gray box indicates the mvpA coding sequence. The translational start site and the direction of translation are indicated by the arrowhead. Probes A and B, described above, are indicated by block boxes. The sizes of restriction fragments are indicated in kilobases. X, XhoI; H,HindIII; B, BarnHI. The mupA gene is single copy and is contained within a 7-kb HindIIIfragment.
particles, at approximately 30 nm in width and 40 nm in length, are also slightly smaller than normal vaults (35 nm X 65 nm). DISCUSSION
Dictyostelium is one of the few species examined to date which expresses two major proteins that are recognized by our anti-rat vault antibody. The observation that this antibody’s affinity for MVPa is much greater than for MVPS suggests that the two speciesof MVP represent separate gene products. This possibility is strongly supported by the continued production of MVPD protein in the M3 cell line in which the mupA locus has been disrupted. In spite of the shared antigenicity, the mvpA and mvpB genes must also be sufficiently unique to account for the inability of mvpA probes to detect mupB message on high stringency Northern blots of M3 RNA. Disruption of the mvpA gene results in viable cells that lack conventional vaults. Thus, MVPa is required for the basic structure of the standard vault particle. In contrast,
46 FIG.4. MVPa protein levels in wild-type and M3 cells. Lanes 1 and 2 contain 20 pg of total cell protein extract from wild-type and M3 cells, respectively. Lane 3 contains 100 ng of wild-type Dictyostelium vaults purified by published procedures (7). Protein was transferred to nitrocellulose and detected with a polyclonal anti-Dictyostelium vault antibody. Note the level of MVPD is reduced in M3 cells compared to wild-type.
characterization of the M3 mvpA- mutants has not revealed any significant difference betweenmutant andwild type cells whengrown in either liquid suspension or on agar plates. They progress through a normal developmental sequenceat a normal rate andform viablespores (data not shown). Chugani et al. (22) have identified vaults at nuclear pore complexes in isolated rat liver nucleiand have speculatedthat vaults might play a role in the transport of materials between the nuclear and cytoplasmic compartments of the cell.Nodifferences were detected in the distribution of [3HH]uridine-radiolabeled total RNA between the nuclear and cytoplasmic compartments of M3 and wild-type Dictyostelium (data not shown), although further experiments are in progress to distinguish if specificmessagelocalizationmight be affectedin MVPadeficient cells. The high degreeof structural andmorphological conservation of the organelle across evolutionarily diversespecies supports the likelihood that vault function is critical to cells. Furthermore, Dictyostelium amoeba, rat liver, and Xenopus oocytes expendcellular resources to maintain abundant levels of the particle reflecting the organelle’s physiological impor-
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FIG. 5. A, Southern blot of M3 genomic DNA. Probe A and B are as described in Fig. 2. Ten pgof M3 genomic DNA was loaded in each lane. Lune I contains BamHIIXhoI-digested M3 genomic DNA hybridized with probes A and B. Lane 2 contains the same digest hybridized with probe B only. Lunes 3 and 4 contain HindIII/XhoI digests of wild-type and M3 genomic DNA, respectively, and were hybridized with probe B. B, restriction map of the mupA- locus in M3 cells. Gray regions represent mupA coding sequence. The translational start sites and the directions of translation are indicated by the arrowheads. Black regionrepresents neomycin resistance cassette. Probes A and B, described in Fig. 2, are indicated byblackboxes. Note the plasmid pMVPaNEO integrated into the mupA locus via single homologous recombination.
FIG. 7. A, electron micrograph of wild-type vaults. Vaults were isolated from wild-type cells by standard procedures and negatively stained with uranyl acetate. Note the characteristic lobular morphology of Dictyostelium vaults. Dictyostelium vaults occur as whole and half structures. Bar, 100 nm. B, electron micrograph of ovoid structures purified from M3 cells. Structures were isolated from M3 cells using standard vault purification procedures and negatively stained with uranyl acetate. Notethe lack of lobular features and the irregular morphology of the M3 structures. Bar, 100 nm.
tance. It seems more likely that in disrupting MVPa protein, we have interrupted normal vault structure butnot function. In fact the presence of an alternate MVPj3-associated structure suggests that vault function may not be impaired in M3 cells. It hasbeen shown that disruptions of single isoforms of multicopy gene families such as annexin VI1 (23) and myosin I heavy chain gene (8) often lack a striking phenotype, and that multiple disruptions may benecessary to generate a more obvious phenotype. Our current goal is to isolate the gene encoding MVP@so as to disrupt it in both a wild type and mvpA- background. Acknowledgments-We are grateful to Dr. Gregory S. Payne (UCLA), Dr. David Knecht (University of Connecticut), and Dr. Meg Titus (Duke University) for helpful discussions and suggestions. REFERENCES 1. Kedersha, N. L., and Rome L. H. (1986)J. Cell BioL 103 699-709 2. Rome, L. H., Kedersha, N. k., and Chugani, D. C. (1991)!"rends CellBioL 1 47-511 ~
1
2
3. KeJe;hi,-N. L., Mi el, M.-C., Bittner, D., and Rome, L.H. (1990)J.Cell Eiol. 110.895-90(1" 4. Kickhoefer V A SearlesR P. Kedersha N. L Garber M. EJohnson, D. L., and &$eL. H. ' 1h3jJ. BWL ckm. 268 7866-7873" 5. Kedersha N. L. Hkuser E., Chugani, D.C., and kome, L. H. (1991)J. Cell Eidl. 112' 225-2& 6. Cohen, S. M.,KAecht, D., Lodish, H. F., and Loomis, W. F. (1986)EMBO J . 5 3361-3366 7. Egelhdff T. Titus T. M. A Manstein D. J Ruppel, K.M., and Spudich, J. A. (i991)ketholrs E&mol. 196,'319-334 8. Manstein, D. J.. Titus, M. A., De Lozanne, A., and Spudich, J. A. (1989)
1.
-
26s mvpA
14. Devereux, J., kaeherli, P., and Smithies, 0.(1984)Nucleic Acids Res. 12,
17s Actin
FIG. 6. mvpA transcript levels in wild-type and M3 lines. Lane I contains 10 pg of total RNA from wild-type cells, and lane 2 contains M3 RNA. The Northern blotwas probed with the full-length MVPa cDNA and chicken actin cDNA (24). Lune 2 had 2.5 times more RNA loaded than lane 1.
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15. S udich J A. (1987)Methods Cell BWL 28 9-100 16. &echt 'D: A Jun J andMatthews L (1990)Deu Genet 11 403-409 17. Knecht: D. A:: a n d k m i s . W. F. (19817)science 236: lO8l~lOd 18. Sambrdok J 'Fritsch E. F. andManiatis T. (1989)hfofecular C h i A Labor&$ M a n u / . 2nd' Ed..Cold Siring . - Harbor Laboratory. y o l d Spring Hirbor NY . 19. Southern E. M. i1975)J. MOLBWl. 98,503-517 20. Jun G 'and Hammer J. A 111 (1990)J. Cell BioL 110,1955-1964 21. 6. F., Tanaka, k. D.;'Svenson K., Wamsley, M., Fogelman, A. M., and &wards P. A. (1987)Mol. Cell. E d . 7 3138-3146 22. Chugani, D. C..'Rome. L. H., and Kedersha, d. L. (1991)J. CeUBioL 115, 45Ha 23. Dorin V Schleicher, M., and Noegel, A. (1991)J. BWL Chem 266, 17sb-it515 24. Cleveland, D. W.,Lopata, M. A., MacDonald, R. J.. Cowan, N. J., Rutter, W. ,J., and Kirschner, M.W. (1980)Cell 20.95-105
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