©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dictyostelium Vaults: Disruption of the Major Proteins Reveals Growth and Morphological Defects and Uncovers a New Associated Protein (*)

Sanjay K. Vasu , Leonard H. Rome (§)

From the (1)Department of Biological Chemistry and the Mental Retardation Research Center, University of California School of Medicine, Los Angeles, California 90024-1737

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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


INTRODUCTION

The vault ribonucleoprotein particle is a cytoplasmic structure identified in a broad spectrum of eukaryotic species(1, 2, 3, 4, 5, 6, 7) . Scanning transmission electron microscopy measurements show rat vaults to have a mass of 12.9 MDa ± 1 MDa(2) . A variety of techniques including cryoelectron microscopy, scanning transmission electron microscopy, and negative staining have been used to determine the particle's dimensions. Although values vary depending on the technique employed, the latter two procedures show rat vaults to have dimensions of 35 60 nm(1, 2, 3) . The ovoid structure resembles a barrel with lobular walls and two centers of mass(1, 2, 3, 4, 5, 6, 7) . When spread on polylysine-coated mica, these two centers of mass split apart, and each barrel half unfolds into a flower-like structure. Each flower is composed of eight rectangular petals linked by short hooks to a central ring(3) .

Although generally cytoplasmic, vault subpopulations have been detected by immunofluorescence at the ruffling edges of spreading rat fibroblasts and along cytoskeletal fibers, suggesting a possible role in motility(2) . Vault association with nuclei has been demonstrated by immunofluorescence on nuclei isolated from rat liver and Dictyostelium discoideum amoeba. The association appears to be specific as it survives washes with 0.5 M NaCl and 1% Triton X-100(7) . Immunogold staining of isolated rat liver nuclei reveals vaults at or near nuclear pore complexes(7) . Although the specific function of vaults is unknown, the 8-fold symmetry, size, dimensions, and immunolocalization suggest a possible function for vaults as the transiently associated central plug/transporter of the nuclear pore complex(3, 4, 7) . Certainly, the conservation of vault morphology between organisms as diverse as rat and Dictyostelium indicates an important and ubiquitous cellular role.

Vaults have been successfully purified from a number of species and characterized most extensively in rat and Dictyostelium(1, 2) . The rat particle includes a 141-base pair RNA species that we have designated vRNA()(1, 5) . vRNA has been shown to be an integral component of the particle as it is protected by the particle from RNase digestion(5) . Isolation and characterization of vRNAs from various organisms including rat and bullfrog have revealed conserved sequences. Moreover, the strong conservation of the predicted secondary structure between the vRNAs suggests that the folded configuration may be important for vRNA function(5) . Although the vault particle affords protection to the vRNA from ribonucleases, it can be digested by prolonged treatment with high ribonuclease activity. Removal of the vRNA in intact vaults does not interfere with vault morphology (determined by electron microscopy) or mobility on nonsieving agarose gels, indicating that the vRNA is not required for the maintenance of vault structure(1) . The Dictyostelium equivalent of the rat vRNA has not yet been identified as purification of Dictyostelium vaults requires treatment with RNase A at levels that preclude survival of an associated RNA.

Dictyostelium vault proteins separate on SDS-PAGE into two bands migrating at 94 and 92 kDa. Since other eukaryotic organisms display only a single major vault protein (Mvp) species, the Dictyostelium doublet was initially considered likely to be the product of differential processing or modification of a single gene product. Subsequent investigation revealed two unique major proteins, MvpA (94 kDa) and MvpB (92 kDa)(6) . Previously, we reported the isolation of a cDNA encoding the MvpA protein(6) . Comparison of MvpA to sequences in GenBank at the DNA and amino acid levels showed MvpA to be unique. MvpA contains none of the known RNA-binding domains and appears to play a predominantly structural role in the particle. Disruption of the gene encoding MvpA is not a lethal event. The resulting mvpA cell line, M3, shows no obvious abnormalities in either growth or development under optimal culture conditions. However, vaults purified from M3 cells lack the characteristic barrel-shaped morphology of normal structures(6) . From this result, we concluded that although MvpA is required for normal vault structure, its presence is probably dispensable for vault function.

In this study, we report the isolation of a cDNA encoding the 92-kDa major vault protein, MvpB. Screens of GenBank at the protein and DNA levels show MvpB's similarity to MvpA(6) , the 104-kDa rat MVP(8) , and a partial human cDNA sequence isolated as an expressed sequence tag. We have now disrupted the gene encoding MvpB in both wild-type and mvpA genetic backgrounds. Characterization of these Mvp-negative lines reveals a growth defect phenotype under suboptimal culture conditions. We also identify a novel 92-kDa protein that copurifies with the ovoid structures isolated from the mvpAmvpB double mutant line. Unlike MvpB, this 92-kDa protein is only poorly recognized by the currently available anti-Dictyostelium vault antibody. We believe that this protein represents a novel vault protein and are currently characterizing it and investigating its role in vault structure and function.


MATERIALS AND METHODS

cDNA Isolation, Sequencing, and Blotting

An M3 Dictyostelium cDNA expression library was constructed in -ZAP (Stratagene, La Jolla, CA). Poly(A) mRNA was prepared from M3 cells (6) and submitted to the UCLA Mental Retardation Research Center Core Facility for library construction. The library was screened using polyclonal anti-Dictyostelium vault antiserum. Plasmid clones were purified away from phage suspension according to the manufacturer's instructions. DNA sequence analysis was performed as described(9) . Protein and nucleic acid blotting analyses were performed as described(6) .

Dictyostelium Culture and Electroporation

The haploid strain JH010 was a gift from Dr. Richard Firtel (University of California at San Diego). The relevant genotype of JH010 is mvpA mvpB thy-1. The strains M6A, M7AB, and M12B were constructed and maintained using previously described methods(6, 10) . HL-5 and SM/5 liquid culture media were prepared as described(11, 12, 13) .

Plasmid Constructs

All enzymes were purchased from Pharmacia Biotech Inc. or Promega. An MvpB cDNA isolate, p8A1, missing 150 base pairs from the 5`-end was excised from -ZAP in Bluescript according to the manufacturer's instructions (Stratagene). The plasmid pGEM-25, containing the Dictyostelium thy-1 gene, was a gift of Dr. Wolfgang Nellen (Max-Planck-Institut fur Biochemie). The intact thy-1 gene was isolated from pGEM-25 as a 3.8-kb HindIII fragment and blunted with Klenow fragment DNA polymerase I. p8A1 was digested with EcoRV, eliminating all MvpB coding sequence except for 1.5 kb from the 5`-end. The blunted thy-1 gene was inserted into EcoRV-digested p8A1. The resulting plasmid, pMvpBTHY, consists of only the truncated 5` 1.5 kb of MvpB coding sequence followed by the thy-1 gene in Bluescript pSK.


RESULTS

Isolation of the MvpB cDNA

To facilitate isolation of an MvpB cDNA, a cDNA library was constructed from the mvpA M3 strain (see ``Materials and Methods''). A positive clone was isolated by immunoscreening the library with a polyclonal anti-Dictyostelium vault antibody. The clone was sequenced and found to share 73% identity at the nucleic acid level with MvpA cDNA. The cDNA contains a full-length open reading frame that encodes a protein with 60% identity at the amino acid level to MvpA (Fig. 1). The similarity to MvpA and the rat MVP throughout the entire sequence confirms this cDNA as MvpB. MvpB shares 50% identity with the 104-kDa rat MVP(8) . Blocks of amino acid sequence identities previously shown to be conserved between MvpA and the rat MVP are maintained in MvpB.


Figure 1: Amino acid sequence comparison between MvpB and other known vault proteins. cDNAs encoding MvpA (dMvpA) and MvpB (dMvpB) from Dictyostelium and the rat MVP (rMVP) were translated, and the full-length predicted amino acid sequences were aligned using University of Wisconsin Genetics Computer Group software (14). Asterisks indicate identities. Comparison shows 60% identity between all three proteins. Numbers designate residue position relative to the predicted amino-terminal methionine.



As expected, searches of GenBank identified MvpA and the rat MVP. However, additional searches using the rat MVP revealed three partial human cDNA sequences, each sharing 85% identity with the rat MVP. These related sequences, isolated as expressed sequence tags, presumably represent a single cDNA clone for the human major vault protein.

As is the case with MvpA, MvpB contains no motifs or domains that might give a clue as to the protein's function. Analysis of the translated cDNA using the Motif algorithm (14) revealed only potential phosphorylation and glycosylation sites. However, the residues that compose these sites include five or fewer amino acids and would be expected to occur randomly in many unmodified proteins. Furthermore, vault proteins in the rat hepatoma cell line H4 are not phosphorylated when metabolically labeled in the presence of [P]orthophosphate(1) . To determine if vault proteins are subject to O-linked glycosylation that occurs on certain cytoplasmic and nuclear proteins, purified Dictyostelium vault samples were separated by SDS-PAGE, blotted to nitrocellulose, and detected with horseradish peroxidase-conjugated wheat germ agglutinin. None of the vault proteins were reactive with the plant lectin and thus appeared to be unmodified with O-linked sugars (data not shown).

Targeted Disruption of mvpB in mvpA and mvpALines

The mvpB gene disruption vector, pMvpBTHY, was electroporated into JH010 cells (mvpA mvpB thy-1), and mvpA mvpBthy-1 transformants were selected in 100-mm tissue culture dishes in the absence of thymidine supplementation(10) . Foci were cultured in 24-well plates and screened by Western analysis for the absence of MvpB. Experience with the mvpA knockouts suggested that <10% of the transformed foci would represent targeted integrations. Moreover, as pMvpBTHY contains 2-fold more thy-1 sequence than mvpB sequence, we expected to see only one mvpB disruption for every two integrations at the mutant thy-1 locus. Western analysis showed that all but two out of 86 thy-1 foci screened contained both the immunoreactive 94- and 92-kDa Mvp bands. One line, M12B, revealed no detectable MvpB protein by Western analysis on crude cell lysate (Fig. 2, lane3).


Figure 2: Detection of vault proteins in wild-type and mvp lines. Total protein from cell lysates was separated by SDS-PAGE, transferred to nitrocellulose, and detected with a 5000-fold dilution of anti-Dictyostelium vault polyclonal antiserum as described previously (6). Lane1, the parental line, JH010; lane2, M6A; lane3, M12B; lane4, M7AB. Twenty µg of protein was loaded in lanes1, 3, and 4; 40 µg was loaded in lane2. The antibody detects only MvpB in M6A and MvpA in M12B lysates. In M7AB, the immunoreactive 80-kDa band presumably represents a truncated MvpB expression product.



The mvpA gene was disrupted in JH010 in order to generate an MvpA-negative line in the same parental line as the mvpB single mutant. This line was subsequently used in the production of an mvpAmvpB double mutant. The plasmid pMvpANEO was electroporated into JH010, and transformants were selected in the presence of G418 (10 µg/ml) as described previously(6) . A candidate line, M6A, which produced no detectable MvpA, was isolated by screening transformants by Western blotting crude lysates (Fig. 2, lane2). No MvpA transcript could be detected (Fig. 3, lane3). In this line, the 7.0-kb HindIII fragment that contains mvpA is split into 3.6- and 5.4-kb fragments (Fig. 4). The mvpB gene in M6A continues to produce a normally sized MvpB protein and transcript ( Fig. 2(lane2) and 3 (lane7)).


Figure 3: Detection of Mvp transcripts in wild-type and mvp lines. Total RNAs from JH010, M6A, M7AB, and M12B cells were separated on a 1% formaldehyde-agarose gel, transferred, and probed with either MvpA or MvpB cDNA. Lanes1 and 5, JH010; lanes2 and 6, M12B; lanes3 and 7, M6A; lanes4 and 8, M7AB. Lanes 1-4 were probed with MvpA cDNA. Lanes 5-8 were probed with MvpB cDNA. Total RNA loaded in all lanes was quantitated by probing with chicken actin cDNA and densitometric scanning (15). All lanes contained 10 µg of RNA, except JH010, which contained 5 µg.




Figure 4: Disruption of mvpA in M6A. Ten µg of JH010 or M6A genomic DNA was digested with HindIII, electrophoresed, transferred, and probed with a 200-base pair internal BglII fragment isolated from the MvpA cDNA. Lane 1, JH010; lane 2, M6A. The 7-kb HindIII fragment shown in lane1 contains the entire mvpA gene in JH010. This fragment is split in M6A into 3.6- and 5.4-kb fragments, revealing the disruption of the gene. Size markers are provided in kilobase pairs.



M6A cells were electroporated with plasmid pMvpBTHY in order to create an mvpAmvpBthy-1 line. Transformants were selected in the absence of thymidine supplementation as described above. Potential positives were screened by Western analysis for the absence of MvpB. One isolate, M7AB, produced no 94-kDa MvpA or 92-kDa MvpB protein as detected by Western analysis of total cell lysate (Fig. 2, lane4). Anti-Dictyostelium vault antiserum did identify a band of 80 kDa in crude M7AB lysate. This immunoreactive band is not present in protein extracts of the parental line, M6A. We do not see this band in M12B cells, which contain an mvpB gene disruption similar to the one in M7AB cells. However, the mvpB disruption in the double mutant line may be subtly different enough to allow low level expression of MvpB coding sequence remaining downstream of the mvpB promoter in M7AB cells (see Fig. 7B). In any case, the M7AB cell line can be considered deficient in MvpB since this immunoreactive protein is smaller than the known Mvps and does not associate with vaults in this cell line (see below). In all three mutants, integration of the gene disruption plasmid recreated Mvp coding sequence downstream of the affected mvp gene. In the case of the mvpB disruptions, this sequence lacks 150 bases of 5`-coding information. We do not discount the remote possibility that these lines might be producing trace amounts of Mvp from recreated downstream coding sequences using fortuitous promoters in the adjacent plasmid. We have, therefore, designated these lines as simply being Mvp-negative. However, with the possible exception of the non-vault-associated 80-kDa band in M7AB, we cannot detect protein products of disrupted mvp genes in mutant cell lysates by Western analysis or proteins associated with purified mutant vaults by silver staining or by Western analysis.


Figure 7: Southern analysis and restriction map of the mvpB gene in M12B and M7AB. Plasmid pMvpBTHY was integrated into the mvpB gene by a single homologous recombination event between the 5` 1.7 kb of the MvpB cDNA and gene. A, genomic Southern blot. Ten µg of DNA was loaded in each lane. Digests were electrophoresed, transferred, and probed with probe 1 (a 1.0-kb HindIII fragment containing the 5`-end of the MvpB cDNA), probe 2 (a 500-base pair KpnI internal fragment of the MvpB cDNA), or probe 3 (a 600-base pair ClaI fragment containing the 3`-end of the cDNA). Lane1, JH010 digested with DraI (D); lane2, M7AB digested with DraI; lanes3 and 4, M12B digested with HindIII (H). Lanes1 and 2 were hybridized with probe 1. The wild-type DraI fragment containing mvpB is split into 4.0- and 2.4-kb fragments in M7AB. Lane3 was hybridized with probe 3, revealing the 3`-end of the disrupted gene in M12B. Lane4 was hybridized with probe 2, indicating insertion of the gene disruption vector into mvpB. B, schematic restriction map of the mvpB gene. Grayboxes represent the mvpB coding sequence. The blackbox represents the thy-1 gene cassette. Plasmid sequence is indicated by the letterv. Blackbars below the gene indicate regions of complementarity between probes and the MvpB coding sequence. Restriction fragment lengths are provided in kilobase pairs.



Restriction Mapping of mvpB Gene Disruptions

The restriction map of the wild-type mvpB gene was established in the parental line, JH010. Digestion of JH010 genomic DNA with a variety of restriction enzymes that do not cut within the MvpB coding sequence generated single bands of even intensity on Southern blots probed with the full-length MvpB cDNA (Fig. 5). This result indicates that mvpB, like mvpA, is a single copy gene. Digestion with DraI and HindIII (Fig. 6A) generates the restriction map shown in Fig. 6B. The entire mvpB coding sequence is contained within a 2.8-kb DraI fragment (Fig. 6A).


Figure 5: Southern analysis of the mvpB gene in JH010. Restriction digests of JH010 genomic DNA (10 µg/lane) with ClaI (lane1), DraI (lane2), EcoRI (lane3), and EcoRI/BamHI (lane4) generate single bands, indicating that mvpB is likely a single copy gene. The Southern blot was probed with a 1.0-kb HindIII fragment containing the 5`-end of the MvpB cDNA. Size markers are shown in kilobase pairs.




Figure 6: Restriction map of the undisrupted mvpB gene. The mvpB gene was mapped in JH010 genomic DNA with respect to DraI and HindIII restriction enzyme sites known to exist in the cDNA. A, genomic Southern blot. Ten µg of DNA was digested with DraI (D; lane1) or HindIII (H; lanes2 and 3). Lanes1 and 2 were hybridized with probe 2. Lane3 was hybridized with probe 1. Probes are indicated in the map in B and are described below. B, restriction map. The graybox represents the mvpB coding region. Blackbars below the gene indicate sites recognized by hybridization probes. Probe 1 is a 1.0-kb HindIII fragment containing the 5`-end of the MvpB cDNA; probe 2 is a 600-base pair EcoRV fragment containing the 3`-end of the MvpB cDNA. Restriction fragment sizes are indicated in kilobase pairs.



MvpB cDNA probes specific to the 5`- and 3`-ends of the MvpB coding sequence were used to map mvpB in the mutants created above. Disruption of mvpB was demonstrated by the loss of the wild-type 2.8-kb DraI fragment (Fig. 7A) in M7AB and M12B genomic DNAs. The coding sequence contained within the DraI fragment is now distributed between an upstream 2.4-kb fragment and a downstream 4.0-kb fragment (Fig. 7A). Digestion with HindIII generates a 7.2-kb fragment consistent with the integration of plasmid pMvpBTHY into mvpB (Fig. 7, A and B).

No MvpB transcript can be detected on high stringency Northern blots of M12B or M7AB total RNA (see Fig. 3). However, at low stringency, a larger (3 kb), weakly hybridizing band appears (data not shown). This could represent cross-hybridization with an RNA related to MvpB. However, as the mvpB promoter faces 1.5 kb of truncated MvpB coding sequence followed by the thy-1 gene cassette, this signal could represent an unstable transcript containing the 5`-end of the MvpB coding sequence fused to noncoding genomic DNA preceding the thy-1 gene. This transcript may result in expression of the amino-terminal half of MvpB, which may represent the 80-kDa protein seen in M7AB crude homogenates.

Identification of a Growth Defect Phenotype in Mvp-negative Lines

The viability of Mvp-negative cells demonstrates that the loss of major vault protein(s) is not a lethal event. However, to evaluate the overall health of these lines, their growth rates were measured and compared with that of the parental line, JH010. Cells were inoculated into the rich broth HL-5 at a density of 1 10 cells/ml, and duplicate flasks of each line were incubated at room temperature, with shaking at 80 rpm. Duplicate aliquots were removed at 24 h intervals, and cells were counted on a hemocytometer. Samples were taken until the cells reached stationary phase. The experiment was repeated, and the results were averaged and plotted (Fig. 8A). The growth curves indicate that, as tested under these optimal axenic growth conditions, all cell lines exhibit roughly equivalent growth kinetics.


Figure 8: Comparison of wild-type and mvp mutant growth rates in HL-5 and SM/5 media. Cultures were seeded with either wild-type (JH010) or mvp mutant lines at a density of 10 cells/ml and grown in the absence of selection. Multiple aliquots were removed at 24-h intervals, and cells were counted. The results were averaged, and cell numbers are plotted versus time in hours. A, growth rates in HL-5. All mutant lines display growth kinetics similar to that of the wild type in rich media. , JH010; , M6A; , M7AB; , M12B. B, growth rates in SM/5. A reduction in mvp mutant growth rates is revealed under conditions of nutritional stress. Mutant growth rates diverge from that of the wild type when cell density reaches 10 cells/ml. Mutant lines reach a final culture density only one-half to one-third that of JH010 cells.



Growth rates of all lines were also evaluated under nutritionally challenging conditions. Although vegetative cells generally divide every 10 h in rich media, their doubling rates slow if the concentration of media additives such as peptone or phosphate drop (12). The growth of the three cell lines was measured in the minimal sporulation medium SM/5(13) . Although parental and mutant lines display slowed growth in the minimal medium, their initial growth rates appear comparable. However, by late log phase, as the cells reach a density of 10 cells/ml, Mvp-negative lines begin to lag behind JH010. The mutant lines reach a final culture density that is, at most, only one-third that of the parent (Fig. 8B). As with wild-type cells, the mutant cultures lyse after reaching stationary phase. Thus, growth in SM/5 reduces doubling times and reveals a minor growth defect in Mvp-negative lines. Assuming vault function is essential to eukaryotic cells, this growth defect suggests that vault function has only been impaired but not abolished in Mvp-negative cells. This defect does not appear to prevent the cells from proceeding through their normal developmental program as plating mutant and wild-type lines on SM/5 agar reveals apparently equivalent abilities to aggregate, form migrating slugs and generate fruiting bodies. Additionally, it would appear that loss of both MvpA and MvpB has no greater impact on vault function than loss of a single Mvp as M7AB growth is comparable to that of M6A and M12B under the conditions of our study.

Characterization of Mutant Vault Morphology

As reported previously, M3 cells contain only MvpB and do not contain characteristic lobular vaults. Attempts to purify vaults from these cells yield an irregularly ovoid structure with dimensions of 30 40 nm (Fig. 9B)(6) . Ovoid vaults were also isolated from M12B cells and examined by negative staining under an electron microscope. The structures present in this MvpB-negative line are indistinguishable from those isolated from M3 (Fig. 9, B and C). These structures are slightly smaller than normal vaults, which have dimensions of 35 65 nm. The lack of topological definition in these structures may result from a loss of structural rigidity due to the absence of one or more Mvps, an idea supported by the fact that the structures are only crudely ovoid, lacking any obvious morphological regularity. In addition, the ovoid structures appear to exist in two forms: a monomeric ovoid and an apparent dimer of two structures separated by a dividing furrow. These monomer and dimer structures are highly reminiscent of the whole and half vaults found in wild-type cells (Fig. 9A)(6) . Similar ovoid structures have been isolated from all mvp mutant lines reported here, including M7AB, which lacks both MvpA and MvpB (Fig. 9D). The complete loss of normal vaults upon the loss of either MvpA or MvpB indicates that both proteins are required to produce and maintain the lobular morphology of normal vaults. The loss of either results in the generation of the amorphous ovoid structure.


Figure 9: Morphological comparison of vaults isolated from wild-type and mvp mutant lines. Vaults were isolated by published procedures, negatively stained with uranyl acetate, and examined by electron microscopy (1). A, normal vaults isolated from JH010; B, ovoid vaults from M3; C, vaults isolated from M12B; D, vaults isolated from M7AB. The loss of either MvpA or MvpB is sufficient to produce the ovoid vault morphology. Ovoid structures persist even in the mvpAmvpB double mutant line, indicating the existence of additional vault components. Note that ovoid structures appear both as monomers and dimers separated by a furrow. Magnification is the same in all four panels. Bar, 100 nm.



We attempted to identify the ovoids as vaults under an electron microscope by use of gold-labeled affinity-purified anti-Dictyostelium vault antibodies. However, the results were unsatisfactory for technical reasons as the available anti-Dictyostelium vault antibody fails to detect even normal vaults under the various staining conditions that were attempted.

The structures from M6A were shown to cofractionate with the MvpB protein peak in the final step of vault purification. Ovoids were isolated using the published Dictyostelium vault purification protocol and fractionated over a 10-50% sucrose step gradient(6) . Electron microscopic analysis demonstrated that the ovoid structures occur in gradient fractions containing MvpB and fractionate differentially from the actin filaments (common contaminants at this stage of purification), suggesting that the structures contain MvpB (data not shown).

Demonstration that ovoid particles represent mutant vaults was achieved in a comparison of ovoid and normal Dictyostelium vault morphologies after various denaturing treatments to which normal rat vaults are impervious(2) . While structural integrity and morphology are unaffected by incubation of rat vaults at room temperature (22 °C), wild-type Dictyostelium vaults subjected to the same treatment convert from the normal lobular morphology to the ovoid form seen in Mvp-negative mutants (Fig. 10, A and B). Conversion is apparently not due to degradation of either MvpA or MvpB since both proteins appear intact as determined by SDS-PAGE analysis of the converted vaults (data not shown). Conversion appears to be an all or none phenomenon as we do not observe partially converted intermediates. This conversion is evidence that the ovoids that copurify with the MvpB protein in M3 and with the MvpA protein in M12B represent what remains of vault structure in these Mvp-negative cells.


Figure 10: Conversion of wild-type vaults into ovoid structures. Normal vaults, exhibiting characteristic lobular morphology, can be converted into ovoid structures upon incubation at room temperature. A, electron micrograph of normal vaults isolated from JH010 and negatively stained with uranyl acetate; B, normal vaults after incubation at 22 °C for 30 min (note that some unconverted structures remain (arrowheads)); C, negatively stained ovoid vaults purified from M3. Bar, 100 nm.



In M7AB, the 80-kDa truncated MvpB protein (Fig. 2, lane4) is not a component of vaults as it does not pellet from a crude cell lysate at 100,000 g with microsomes and vaults (data not shown). However, despite the absence of both full-length MvpA and MvpB, ovoid vaults are still present in this line. These structures can be purified by standard vault purification techniques and are visually indistinguishable from vaults found in mvpA and mvpB lines. Vaults isolated from M7AB cells and analyzed on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels fractionate with a protein that migrates at 92 kDa, approximately the same position as MvpB. Unlike MvpB, this novel 92-kDa protein, MvpC, is only weakly recognized by anti-Dictyostelium vault antiserum on Western blots of purified M7AB vaults (Fig. 11, B and C).


Figure 11: Western analysis of vaults isolated from mvpA and mvpAmvpB cells. Vaults were isolated from M6A and M7AB and fractionated on 10-50% sucrose gradients according to published procedures (1). Gradients were fractionated from the top to the bottom, and fractions were examined by staining with Coomassie Brilliant Blue (A and D) and by Western analysis (B and C). Blots were probed with a 5000-fold dilution of anti-Dictyostelium vault antiserum as described previously (6). A: lanes 1-4, protein peak from M6A vault sucrose gradient. B: Western blot of gel in A. Anti-Dictyostelium vault antiserum detects only MvpB and no MvpA protein in these fractions. The antibody cross-reacts with actin, a consistent contaminant of vault preparations. C: lanes 1-4, Western blot of protein peak from M7AB vault sucrose gradient; lane5, wild-type vaults. A 92-kDa protein copurifies with M7AB ovoid structures. D: Coomassie Brilliant Blue-stained gel of M7AB samples shown in C. Although twice as much protein was loaded in C and D (2 µg/lane) as in A and B (1 µg/lane), the 92-kDa protein's cross-reactivity with anti-Dictyostelium antiserum is much weaker than that of MvpB shown in A and B.




DISCUSSION

Dictyostelium is the only organism (with the possible exception of rabbit) known to contain more than one Mvp. Vault proteins isolated from this organism, designated MvpA and MvpB, migrate as a doublet at 92-94 kDa on SDS-PAGE(2) . The first evidence that the Dictyostelium Mvp doublet represented multiple gene products was provided by the anti-rat polyclonal vault antibody's differential reactivity against MvpA versus MvpB. MvpB lacks epitopes recognized by the anti-rat vault antibody that are present in MvpA. Since the Mvps are apparently unmodified, this result was the first indication that the two Mvps were derived from different genes. The first Mvp cDNA to be isolated maps to a single copy gene, whose disruption eliminates only MvpA transcript and protein. Since these cells continue to produce MvpB, this second Mvp is a unique gene product. The divergence between mvpA and mvpB is demonstrated by the failure of MvpA cDNA probes to detect the MvpB transcript on blots of M3 total RNA. MvpB cDNA probes are insufficiently similar to mvpA gene sequences to allow cross-detection of mvpA on high stringency genomic Southern blots. Subsequent isolation and analysis of the MvpB cDNA revealed MvpB to be only 60% similar to MvpA at the amino acid level. The extent to which the proteins differ outside of the conserved regions suggests that these divergent areas either are not functionally imperative domains or are functionally divergent.

Cell lines lacking either or both MvpA and MvpB show no morphological, developmental, or growth defects under permissive culture conditions. This finding suggests that MvpA and MvpB are not required components of vaults. However, when placed under nutritional stress, mvp lines display a mild growth defect. Thus, vault function in the mutant lines is likely to be at least partially impaired. Losing two Mvps does not seem to increase the magnitude of the growth defect over the loss of a single Mvp. Thus, the impediment to vault function is presumably equivalent in both single and double mvp mutant lines. Whether this impediment is due to loss of Mvp-mediated function or to a reduction in the particle's structural integrity is unclear.

Vaults isolated from mvp lines can be visualized under an electron microscope as ovoid structures that lack any of the symmetry or lobular morphology characteristic of normal vaults. No characteristic lobular vaults were found in either mvpA or mvpB lines by electron microscopy, indicating that both proteins are required for normal vault structure. The absence of discernible nonlobular ovoid vaults in wild-type cells suggests that these structures represent Mvp-deficient particles and not a naturally occurring subclass of vaults composed of a single Mvp species. Demonstration that normal Dictyostelium vaults can be induced by temperature shift to undergo transformation into ovoid structures verifies that these structures represent vaults. In all mutant lines, ovoid vaults fractionate with remaining unaltered Mvps. Surprisingly, ovoid vaults could also be isolated from the mvpAmvpB double mutant line, indicating that MvpA and MvpB are not the sole components of Dictyostelium vaults. The M7AB ovoid vaults copurify with a single 92-kDa protein that is very poorly detected by available anti-Dictyostelium vault antisera. This protein, which we have designated MvpC, appears to lack epitopes conserved between not only MvpA and MvpB of Dictyostelium, but also Mvps of rat and Xenopus (data not shown). The failure of MvpA and MvpB cDNA probes to detect MvpC message on Northern blots of total RNA from mvpA and mvpB cells reflects this divergence. MvpC's structural divergence suggests functional divergence as well. This protein could represent an additional or partially redundant functional element in the Dictyostelium vault. Alternatively, all three Mvps may exhibit partial functional redundancy, and any one Mvp may be able to compensate for the loss of the other two proteins. Finally, it is also possible that MvpC is not normally a component of Dictyostelium vaults and has been induced in the Mvp-negative lines. This idea is supported by the fact that our current anti-Dictyostelium vault antibody, which was made against whole wild-type Dictyostelium vaults, cross-reacts poorly with MvpC.

Isolation of an MvpC cDNA will be the first key step in characterizing this new vault protein as well as in determining its role in vault function through gene disruption experiments in wild-type and mvp mutant lines. Using MvpA and MvpB cDNA probes in low stringency library screens has thus far failed to detect an MvpC clone. The available anti-vault antisera are insufficiently reactive toward MvpC to be useful in immunoscreening procedures. We are currently attempting to generate a polyclonal antibody against MvpC protein purified from M7AB cells. Such antibodies should enable isolation of a cDNA clone from available Dictyostelium expression libraries.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM 38097. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) Z37109.

§
To whom correspondence should be addressed. Tel.: 310-825-0709; Fax: 310-206-5272; E-mail: lrome@biochem.medsch.ucla.edu.

The abbreviations used are: vRNA, vault RNA; PAGE, polyacrylamide gel electrophoresis; Mvp, major vault protein; kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Carol Gray for photographic services and gratefully acknowledge Dr. Gregory Payne for helpful comments and critical reading of this manuscript. We thank Dr. Valerie Kickhoefer for assistance with GenBank searches and for identification of MVP-related human cDNAs (GenBank accession numbers Z18337, Z17891, Z17858, and D29489).


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