From the
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
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
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
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
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
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
[
No MvpB transcript can be
detected on high stringency Northern blots of M12B
The structures from M6A
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
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
Vaults isolated from mvp
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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).
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
genetic backgrounds. Although the mvp
mutant lines are viable, they show that
loss of MvpA and/or MvpB interferes with vault function sufficiently to
impede growth under conditions of nutritional stress. The resulting
mutant cell lines reach stationary phase in suspension culture at
one-third of the density of wild-type cells. Ovoid structures isolated
from mvp
single mutant lines represent what
remains of vaults in these cells. Similar ovoid structures isolated
from the mvpA
mvpB
line copurify with a newly identified protein of 92 kDa (MvpC),
which lacks cross-reactivity with currently available anti-vault
antibodies. Our results indicate that the major vault proteins are
necessary for optimal cell growth in Dictyostelium and reveal
an unanticipated complexity in vault composition.
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) .
(
)(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.
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.
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 mvpA
mvpB
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.
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
,
M7A
B
, 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
.
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.
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
mvpA
The mvpB gene
disruption vector, pMvpBTHY, was electroporated into JH010 cells (mvpA mvpB thy-1Lines
), and mvpA mvpB
thy-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,
M7A
B
. 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 M7A
B
, 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 mvpA
mvpB
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
, M7A
B
, 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,
M7A
B
. 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 mvpA
mvpB
thy-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,
M7A
B
, 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
M7A
B
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 M7A
B
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
M7A
B
cells (see Fig. 7B). In any case, the
M7A
B
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
M7A
B
, 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
M7A
B
. 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,
M7A
B
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 M7A
B
. 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
M7A
B
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).
or
M7A
B
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 M7A
B
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
;
,
M7A
B
;
,
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
M7A
B
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 M7A
B
, 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
M7A
B
. The loss of either MvpA or
MvpB is sufficient to produce the ovoid vault morphology. Ovoid
structures persist even in the mvpA
mvpB
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.
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).
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
M7A
B
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 M7A
B
vaults (Fig. 11, B and C).
Figure 11:
Western analysis of vaults isolated from mvpA and mvpA
mvpB
cells. Vaults were isolated from
M6A
and M7A
B
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 M7A
B
vault
sucrose gradient; lane5, wild-type vaults. A 92-kDa
protein copurifies with M7A
B
ovoid
structures. D: Coomassie Brilliant Blue-stained gel of
M7A
B
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.
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.
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 mvpA
mvpB
double
mutant line, indicating that MvpA and MvpB are not the sole components
of Dictyostelium vaults. The
M7A
B
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.
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
M7A
B
cells. Such antibodies should
enable isolation of a cDNA clone from available Dictyostelium expression libraries.
/EMBL Data Bank with accession number(s)
Z37109.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.