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First published online March 2, 2007
Journal of Experimental Biology 210, 946-955 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001800
The major vault protein is related to the toxic anion resistance protein (TelA) family
1 Department of Molecular Biosciences, University of Kansas, Lawrence, KS
66045, USA
2 Bioinformatics Core Facility, University of Kansas, Lawrence, KS 66045,
USA
3 Molecular Graphics and Modeling Laboratory, University of Kansas,
Lawrence, KS 66045, USA
* Author for correspondence (e-mail: ksupre{at}ku.edu)
Accepted 10 January 2007
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Summary |
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Key words: protein structure, ribonucleoproteins, terratogens, toxins fluorescent antibody technique, indirect and computational molecular biology
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). To
date, no prokaryote relatives of the vault particle have been identified. The
barrel-shaped vault complex comprises the major vault protein (MVP),
telomerase-associated protein-1 (TEP1) and the vault poly(ADP-ribose)
polymerase (VPARP), and an untranslated small RNA
(Suprenant, 2002). The major
vault protein (MVP) comprises 75% of the mass of the vault particle and
expression of MVP in baculovirus-infected insect cells is sufficient to
self-assemble into a vault-like particle with the same overall shape and
dimensions of the native vault structure
(Stephen et al., 2001).
Surprisingly, these vault shells are very dynamic and are capable of
`breathing' to allow the incorporation of TEP1, VPARP and potential cargo
molecules (Poderycki et al.,
2006).
The function of vaults remains elusive. High levels of vaults are
correlated with a poor prognosis in certain cancers and with some multi-drug
resistant (MDR) cancer cell lines
(Scheffer et al., 2000).
Although most vaults reside in the cytosol, a small but detectable fraction
(
5%) is associated with the nucleus and it has been suggested that vaults
may be involved in nucleocytoplasmic transport
(Chugani et al., 1993;
Hamill and Suprenant, 1997;
Slesina et al., 2005;
Stewart et al., 2005;
van Zon et al., 2006). MVP may
act as a scaffolding protein for the protein tyrosine phosphatase, SHP-2 and
activated extracellular-regulated kinases (ERKs)
(Kolli et al., 2004). In
addition to interacting with the epidermal growth factor signaling pathways,
MVP expression is upregulated through the JAK/STAT signaling pathway
(Steiner et al., 2006).
From molecular analyses and high-resolution cryoelectron microscopy, we
know that vault composition and structure are highly conserved
(Kedersha et al., 1990;
Kong et al., 1999;
Mikyas et al., 2004;
Stewart et al., 2005). The
primary sequence of MVP comprises seven short repeats of 55 amino acids
at the amino terminus, a central region and a coiled-coil interaction domain
at the carboxyl terminus (van Zon et al.,
2002), as depicted schematically in
Fig. 1A. The repeat unit (aa
26–401) is highly conserved across eukaryotes and forms the basis for a
characteristic protein sequence domain. The solution NMR structure of a
two-repeat domain of the MVP reveals that the walls of the central barrel are
built from repeating units of a unique three-stranded anti-parallel sheet
(Kozlov et al., 2006). This
structural repeat is the same length as the sequence repeat but is shifted by
half of a sequence repeat (26 amino acids). The coiled-coil domain at the
C terminus is thought to align the MVPs at the vault cap
(Kozlov et al., 2006;
Mikyas et al., 2004;
van Zon et al., 2002).
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). The second search iteration yielded the tellurite
resistance protein (TelA) from Rhodobacter sphaeroides (AF019377) as
a plausible match (homologous alignment with E=6e–15). Based on the
results of our initial BLAST search for homologs to the MVP repeat unit in
which TelA was identified, we performed a reciprocal search based on the
sequence of tellurite resistance protein, retrieving the eukaryotic MVP in a
second round of iteration (E<3e–8). Further analysis of the
eukaryotic MVP domain in common with the toxic ion resistance proteins
revealed that the highest degree of similarity was found in a large internal
repeat (residues 103–221; 156–276). An iterative search with this
internal repeat (103–221) also identified the tellurite resistance
protein (E=2e–14).
Having identified internally consistent evidence for homology between the
eukaryote MVP repeat and TelA, a sequence alignment of two was carried out
using the ClustalW program (Thompson et
al., 1994) with the BLOSUM-30 substitution matrices (permitting
both positive and negative match scoring)
(Henikoff and Henikoff, 1992),
a gap-opening penalty of 10 and a gap-extension penalty of 0.1. The portion of
the sequence corresponding to the MVP domains 3 and 4 resolved via
NMR (Kozlov et al., 2006)
yielded a net identity score of 31% relative to TelA, and a similarity score
of 56%. These scores would normally suggest a probable homologous (potential
superfamilial) relationship and a reasonable probability of a conserved domain
3/4 fold, but further scrutiny of the computed alignment relative to the
three-dimensional structure revealed unphysical gaps within key MVP beta
strands that would preclude common folding. Intuitive manipulation of the
alignment to preferentially shrink gaps within strands at the expense of known
coil regions, however, led to formulation of an alternative sequence
alignment, reported in Fig. 1B,
that retains 30% TelA identity and 55% similarity relative to the domain 3/4
region of MVP. This alignment meets the basic minimum criterion for meaningful
homology modeling (30% sequence identity), and appears to be physically
reasonable, thus we used it as the basis for comparative structure prediction
for the portion of TelA in alignment with MVP domains 3 and 4. The TelA model
was then constructed via the Modeller program
(Marti-Renom et al., 2000).
Default restraint settings were retained, and rigorous simulated annealing
steps using the Chemistry at HARvard Molecular Mechanics (CHARMM) force field
and charges (MacKerell, Jr et al.,
1998) were employed in order to permit reasonable relaxation of
the 12-residue TelA loop (MQSLPSIRLVQE), for which no MVP template residues
exist. Specifically, five simulated annealing cycles of 4.4 ps stepwise
warming (0 K
150 K250 K400 K700 K1000 K) followed
by 19.2 ps stepwise cooling (1000 K800 K600 K500 K400
K300 K) were performed. The resulting structure was then examined for
unrealistic contacts and geometry via the ProCheck program
(Laskowski et al., 1993).
Further structural analysis on the proteins was carried out using the SYBYL
suite of programs (Tripos,
2006).
Antibodies
The primary antibodies used in this study were: anti-ß-tubulin
antibody Tub2.1 (Sigma-Aldrich, St Louis, MO, USA), anti-MVP antibody LDQN
(Eichenmuller et al., 2003),
anti-VPARP antibody ACE (a kind gift of Valerie Kickhoefer, David Geffen
School of Medicine at UCLA, Los Angeles, CA, USA)
(Kickhoefer et al., 1999),
anti-PABP antibody 10E10 (a kind gift of Gideon Dreyfuss, University of
Pennsylvania, School of Medicine, Philadelphia, PA, USA)
(Gorlach et al., 1994),
anti-HnRNPA2 antibody EF67 (a kind gift of William Rigby, Dartmouth Medical
School, Lebanon, NH, USA) (Nichols et al.,
2000), anti-TIA-1 3E6 (a kind gift of Nancy Kedersha, Brigham and
Women's Hospital, Boston, MA, USA) (Taupin
et al., 1995) and anti-ubiquitin antibody FK-2 (StressGen
Biotechnologies, Ann Arbor, MI, USA).
Cell culture
HeLa cervical cancer cells (ATTC CCL-2) and human umbilical vascular
endothelial cells (HUVEC; ATCC CRL-1730), were grown in T-75 tissue culture
flasks in Dubecco's Modified Eagle's Medium (DMEM)/Hams F12 supplemented with
2 mmol l–1 L-glutamine, 10% fetal bovine serum, 100 units
ml–1 penicillin and 100 µg ml–1
streptomycin. HeLa cells used in MVP siRNA knockdown assays were cultured in
DMEM containing 4 g l–1 glucose and supplemented with 4 mmol
l–1 glutamine, 10% fetal bovine serum (FBS), 500 units
ml–1 penicillin, 0.1 mg ml–1 streptomycin
and 0.01 mg ml–1 of Tylosin. HUVEC culture medium
additionally contained 15 µg ml–1 endothelial cell growth
supplement (Biomedical Technologies, Inc., Stoughton, MA, USA) and 100 µg
ml–1 heparin. The culture flasks for the HUVECs were coated
with type-1 rat-tail collagen. All cells were maintained in an atmosphere with
5% CO2 at 37°C, unless otherwise noted.
Immunofluorescence microscopy
Adherent cells were detached from the flasks by a 5 min incubation
(37°C) with a trypsin-EDTA solution (0.01% crystalline trypsin, 0.1% EDTA
in divalent-cation-free Dulbecco's PBS). The cells were washed once with
medium containing 5% FBS, diluted, seeded onto coverslips and grown for 24 h
at 37°C. Cells were treated with either potassium tellurite
(K2TeO3), sodium selenite
(Na2SeO3), sodium selenate
(Na2SeO4), sodium arsenite
(Na3AsO3), sodium arsenate
(Na3AsO4) or sodium orthovandate
(Na3VO4), as described in the Results, and fixed with
either –20°C methanol for 5–7 min or 2–4%
paraformaldehyde for 15 min. No differences were observed in the staining
patterns for the anti-vault antibodies with either fixation procedure so
methanol fixation was used throughout the studies reported. The cells were
rinsed three times with PBS and incubated with the primary antibodies for 60
min at 37°C. After rinsing the coverslips four times (10 min each rinse)
with PBS, the cells were incubated with either Cy3- or Cy2-conjugated
secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA,
USA). The coverslips were rinsed three times with PBS and once with 300 nmol
l–1 4',6-diamidino-2-phenylindole (DAPI) in PBS to
counterstain the nuclei. The coverslips were mounted in 90% glycerol and 10%
PBS, pH 9.0 containing 4% (w/v) n-propyl gallate. Images were
acquired by using a Nikon Optiphot microscope equipped with a Zeiss 63X, 1.3
NA objective lens and an Orca ER cooled CCD camera (Hamamatsu, Bridgewater,
NJ, USA). Image acquisition was controlled by OpenLab software (Improvision,
Inc., Lexington, MA, USA) and compiled using Adobe Photoshop.
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) indicates
that both the MVP repeat segment and its putative TelA homolog display
extensive and reasonably strong similarity (greater than 60%) with S-layer
paracrystalline bacterial surface coatings. These findings strongly suggest
that aspects of mammalian MVP are more closely related to a variety of
prokaryotic proteins, strongly bolstering the plausibility for a common
ancestry between the eukaryotic MVP and TelA.
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)
suggests that our predicted TelA structure may be under less strain (only nine
bad atomic contacts flagged) than the MVP template itself (20 bad contacts) on
which the TelA model was based, perhaps due to explicit solvent effects on the
latter NMR structure. Otherwise, ProCheck analysis provided a comparable
assessment of the physical soundness of both structures, determining an
average G-factor score (accounting for all torsions, bond angles and bond
distances) of –0.50 for MVP and –0.48 for TelA, both of which are
well within the acceptable range. An indication of the relative size, shape and surface charge distribution of our predicted TelA model and its MVP template is given in Fig. 2B. Given the fact that the portion of the TelA sequence corresponding to domains 3,4 of MVP resolved via NMR is longer by 22 amino acids (135 vs 113), it is not surprising that the predicted TelA volume (28 212 Å3) and surface area (9083 Å2) are somewhat larger than that computed for MVP (24 096 Å3 and 7954 Å2, respectively). This is reflected in a marginally larger appearance in the rendered versions in Fig. 2B. The shape of the two proteins, however, is very similar, with the only major difference between the two being an additional lobe just below the upper right corner of the TelA structure, corresponding to the aforementioned unmatched 12 residues between the 4th and 5th strands of the sheet structure. This extended coil is located mostly on the surface of the TelA structure, which suggests that it is unlikely to significantly perturb the basic fold of the protein relative to MVP, but may yield modest differences in protein-protein associative tendencies, environmental preferences, and functionality. Beyond this, however, the two systems have very similar electrostatic/hydrophobic profiles, with polar residues constituting 41.7% of the MVP and 40.5% of the TelA surface areas. It is interesting to note, however, that while MVP has only a modest difference in the relative surface areas of cationic and anionic residues (1264 and 1185 Å2, respectively), the ratio is significantly more slanted toward surface cations in the case of TelA (1531 vs 1047 Å2).
MVP aggregates form in response to tellurite
We investigated whether MVP abundance or cellular location changed in
response to tellurite treatment. Previously, we generated an antipeptide
antibody (anti-LDQN), against conserved sequences within the amino terminus of
the MVP, and showed that MVP localizes predominantly to the cytosol in a
punctate staining pattern (Eichenmuller et
al., 2003). In the HeLa cytosol, MVP is concentrated near the
nucleus where the cell is the thickest
(Fig. 3A). In addition,
unidentified intranuclear speckles are also observed with this anti-MVP
antibody. As you move outwards towards the cell periphery the number of MVP
puncta diminishes. A similar staining pattern has been described with other
MVP antibodies (Kedersha et al.,
1990; Kickhoefer et al.,
1999; van Zon et al.,
2003). When potassium tellurite (K2TeO3) is
added to the culture medium, MVP is no longer concentrated near the nucleus.
In contrast, MVP is found in large, irregularly shaped cytoplasmic aggregates.
In some cases, the aggregates are as large as 7 µm in diameter. The MVP
aggregates are found predominantly at the cell margins where the cell makes
contact with the substrate. Aggregate formation is temperature sensitive and
can occur in 15 min or less and at tellurite concentrations as low as 5
µm.
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As mentioned above, nearly all the MVP in the cell is assembled into a
vault particle (Kickhoefer et al.,
1999; Siva et al.,
2001). Therefore it is likely that vault particles move to the
cell margins in response to tellurite treatment. To be certain that this is
indeed the case, tellurite-treated cells were immunostained with a polyclonal
antibody against the vault-associated poly(ADP-ribose) polymerase (VPARP).
VPARP catalytically adds ADP-ribose polymers to itself and MVP and is located
in three stave-like bands on the inside of the vault barrel
(Kickhoefer et al., 1999;
Mikyas et al., 2004). In HeLa
cells, anti-VPARP antibodies stain peri-nuclear cytoplasmic puncta as well as
intranuclear speckles (Kickhoefer et al.,
1999) (Fig. 3F). In
the presence of tellurite, VPARP staining occurs in large aggregates at the
cell periphery (Fig. 3G). Since
MVP and VPARP are integral components of vaults, it is likely that the
peripheral accumulations of VPARP and MVP occur together in an intact vault
particle. We refer to these large vault aggregates as vaultosomes.
Vaultosomes form at the cell margins in non-transformed endothelial cells
Since HeLa cells are derived from an aggressive glandular cervical cancer,
it is important to determine whether vaults respond to tellurite treatment in
a non-transformed cell line. For these experiments, we examined vault location
in HUVEC cells, an endothelial cell line derived from normal human umbilical
cord. In untreated HUVEC cells, MVP is concentrated in the cytosol near the
nucleus in a pattern that is indistinguishable from that seen in the aneuploid
HeLa cell line (Fig. 4A). Upon
tellurite exposure, MVP staining appears to be concentrated at the cell
periphery in irregularly shaped patches or aggregates
(Fig. 4B). Thus, vaults respond
to tellurite in a similar manner in these two different human cell lines.
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). HeLa cells were exposed to 0.5 mmol l–1
tellurite for 30 min, washed with fresh medium and allowed to recover in
tellurite-free medium overnight. Over a24 h period, the cell number had
doubled in both the tellurite-treated and untreated cultures (compare
Fig. 4E with H). In addition,
the vaultosomes were no longer present at the cell margins. The MVP staining
pattern in the tellurite-treated and recovered cells was identical to the
untreated cells (compare Fig. 4C with
E). These experiments lead us to conclude that vaultosome
formation is a dynamic and reversible process.
Tellurite does not depolymerize the microtubule cytoskeleton
Previous studies have shown that vaults bind directly to microtubules
in vivo and in vitro
(Eichenmuller et al., 2003;
Hamill and Suprenant, 1997).
To be certain that vault aggregation at the cell margin was not dependent upon
microtubule reorganization or depolymerization, the cells shown in
Fig. 4 were double stained with
antibodies against tubulin and MVP. In untreated HeLa and HUVEC cells, the
microtubules extend radially from the mitotic organizing center near the
nucleus to the cell periphery. Exposure to tellurite did not have a great
effect on the organization of the HeLa cell microtubules (compare
Fig. 4F with G). In the HUVECs,
the microtubule array also extends radially from the nucleus to the cell
surface although the microtubules are somewhat collapsed in regions where the
cells lost their attachment to the coverslips. Tellurite appears to be more
toxic to the HUVECs. Even so, the vaults were still aggregated at the cell
periphery (Fig. 4B). These
experiments indicate that a catastrophic change in the microtubule array was
not responsible for the tellurite-induced movement of vaults to the cell
margin.
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Vaultosomes do not form during a general stress response. To determine
whether vault aggregation was a general stress response, HeLa cells were
heat-shocked (44°C, 60 min) or UV irradiated (100 µJ) and the location
of the MVP was monitored by immunofluorescence. Similar to a previous study
(Kickhoefer et al., 1999), no
changes in the distribution of the MVP were observed with either treatment
(data not shown).
Vaultosomes form independently of other ribonucleoprotein complexes
Arsenite is known to inhibit protein synthesis and to accumulate
untranslated RNAs into dense stress granules
(Anderson and Kedersha, 2002).
Since the vaultosomes also formed under toxic ion stress, we reasoned that the
vaultosomes might be large enough to harbor the stress granules. To examine
whether stress granules were associated with vaultosomes, we treated cells
with arsenite and immunostained the cells with antibodies against vaults and
the RNA-binding protein TIA-1, a robust marker of mammalian stress granules
(Kedersha et al., 1999).
Arsenite treatment induced the formation of vaultosomes and the smaller stress
granules; however, neither particle colocalized with each other in the cell
(Fig. 6). Furthermore,
vaultosome formation was not affected by drugs (emetine, puromycin) that
affect the dynamic equilibrium of RNAs in the stress granules
(Kedersha et al., 2000) (data
not shown). These results indicate that vaultosomes form independently of
stress granules.
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To examine whether other RNPs were associated with the vaultosomes, we
treated cells with arsenite and immunostained the cells with antibodies
against either the mRNA polyA-binding proteins-I (PABP-I) or the heterogeneous
nuclear ribonucleoprotein A2, a protein implicated in translational
regulation, RNA processing and RNA transport
(Dreyfuss et al., 1993;
Hamilton et al., 1999;
Kwon et al., 1999). Neither
RNA binding protein, PABP-I or hnRNP A2 appear to colocalize to a significant
extent with the vaults in untreated cells or arsenite-induced vaultosomes
(data not shown).
Vaultosomes are not aggresome-like
In response to proteosome inhibition, aggregates of ubiquinated proteins
and proteosome subunits will accumulate around the microtubule organizing
center of HeLa cells (Wojcik et al.,
1996). These `aggresomes' form when the synthesis of misfolded
proteins exceeds the capacity of the chaperones and the proteosomes
(Johnston et al., 1998). We
examined whether vaultosomes colocalized with ubiquinated proteins in the
aggresomes when cells are treated with the proteosome inhibitor,
N-acetyl-Leu-Leu-Norleucinal (ALLN). Fig.
7 shows that aggregates of ubiquinated proteins are found near the
nucleus of the HeLa cells after treatment with ALLN. We were surprised to find
that vaults also aggregated when the HeLa cells were treated with ALLN,
however the vaultosomes did not colocalize with the ubiquinated proteins in
the aggresomes. These results show that vaultosomes form in response to
proteosome inhibition; however, vaultosomes do not colocalize with the
aggresomes.
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). In humans, elemental tellurium is very toxic and induces
both chronic and acute toxicity, with death being the most extreme outcome.
Although the toxic metalloid tellurium is relatively rare in the natural
environment, several electrical, metallurgical and chemical manufacturing
applications generate elevated levels of tellurium in the environment.
The toxicology of tellurium compounds on humans and other animals has been
studied in detail, but the basic mechanism(s) of cellular toxicity is not
known. For example, one of the earliest documented biological effects of
tellurium poisoning is the demyelination of the sciatic nerve due to the
inhibition of cholesterol biosynthesis and the accumulation of squalene in
Schwann cells (Harry et al.,
1989; Lampert et al.,
1970). In bacteria, the toxicity of the soluble oxyanion
tellurite, TeO –23, is thought to be due to its
strong oxidizing effects on many enzymatic processes
(Summers and Jacoby, 1977) and
its ability to replace sulfur in metabolic reactions
(Turner et al., 1995).
Tellurite resistance in bacteria
Although bacteria rarely come into contact with tellurium, with the
exception of highly polluted environments, there are several different and
seemingly unrelated tellurite resistance (TeR) determinants that
are found both on plasmids and in the genomes of unrelated bacteria
(Taylor, 1999). In general,
bacterial TeR does not involve active efflux or reduced uptake of
tellurium compounds, but rather the conversion of tellurite to a form that is
less toxic to the bacteria. Some determinants involve basic cysteine
metabolism (O'Gara et al.,
1997) or methyl group transfer
(Cournoyer et al., 1998) to
detoxify tellurite. In E. coli, a basal level of tellurite resistance
is brought about by nitrate reductase enzymes that are able to reduce
tellurite and selenate at the cell membrane
(Avazeri et al., 1997). In
addition, the thiol:redox buffering system (glutathione and thioredoxin)
appears to play an important role. One of the targets for tellurite toxicity
in E. coli as well as erythrocyes is glutathione
(Deuticke et al., 1992;
Turner et al., 2001;
Young et al., 1981). However,
at this time there is no unifying theory of tellurite resistance. In this
regard, tellurite resistance is thought to be a secondary effect of a normal
metabolic or stress response pathway
(Taylor, 1999).
The photosynthetic bacteria, Rhodobacter sphaeroides strain 2.4.1,
has a surprising metabolic repertoire that includes anaerobic photosynthesis,
aerobic and anaerobic respiration, as well as nitrogen and carbon fixation.
R. sphaeroides survives in high levels of rare-earth oxides and
oxyanions such as tellurite and selenite
(Moore and Kaplan, 1992).
TeR in R. sphaeroides 2.4. is associated with two loci,
trgAB and telA (O'Gara
et al., 1997). The trgAB genes confer TeR when
introduced into the related bacterium Paracoccus denitrificans. In
addition, a TelA null strain shows a partial decrease in resistance when
compared to a wild-type strain. The mechanism of TrgAB and TelA associated
resistance remains unknown.
The TelA protein family
The toxic anion resistance protein (TelA) family consists of several
prokaryotic TelA-like proteins including the TelA protein of R.
sphaeroides, D. radiodurans, B. subtilis and Y. pestis
(O'Gara et al., 1997), the KIA
protein associated with plasmid fertility inhibition
(Whelan et al., 1995) and the
KlaA protein from the methanomicrobe, Methanosarcina acetivorans.
In this report, we have shown that the eukaryotic major vault protein also
shares significant sequence similarity with the TelA protein of
Rhodobacter sphaeroides, and make a reasonable argument that the two
share a common fold, spatial size and shape, and electrostatic profile. A
solution NMR structure reveals a unique, novel fold consisting of a
three-stranded antiparallel ß-sheet
(Kozlov et al., 2006). Our
molecular modeling of the bacterial TelA protein reveals a common fold.
Further evidence for a common MVP and TelA ancestry was obtained when it was
shown that the MVP repeat segment and the TelA protein display greater than
60% similarity with S-layer paracrystalline bacterial surface coatings. These
results indicate that the TelA protein may be a distant relative of the major
vault protein.
In bacterial cells, it has been speculated that tellurite resistance is not
the primary property of any of the unrelated tellurite resistance determinants
(Taylor, 1999). The lack of
similarity between any of the proteins associated with tellurite resistance
has confounded their functional analysis. In addition, most bacteria rarely
encounter high levels of tellurite in their environment so it is difficult to
understand why so many bacteria maintain these genes in their genomes and on
plasmids. If the primary function of the TeR proteins is not to
confer tellurite resistance why have they been conserved amongst so many
divergent bacteria?
An intriguing hypothesis was put forth
(Taylor, 1999) that the
primary role of the TeR determinants might be to protect the
bacterium from mammalian host defenses. Many pathogenic bacteria are resistant
to tellurite and one or more TeR determinants have been identified
in Y. pestis, M. tuberculosis and N. meningitidis, for
example. In Taylor's model, the products of the TeR genes are able
to counterattack toxic substances produced by macrophages and other human
cellular defenses. It is curious that mammalian vaults are very abundant in
macrophages and in the dendritic cells of the immune system
(Mossink et al., 2003;
Mossink et al., 2002;
Schroeijers et al., 2002). It
is intriguing to speculate that macrophages inherited a tellurite-resistant
gene from an ancestral pathogen and that vaults are an unrealized component of
a metabolic pathway that originated in bacteria.
Vault function?
Movement of vaults to the cell surface in response to tellurite suggests
that there is a link between MVP and the TelA protein. In response to a
sublethal dose of K2TeO3, large vault aggregates appear
at the cell margins. Aggregate formation is dynamic and reversible and occurs
in normal cells grown in culture as well as cancer cell lines. Vaultosomes
form independently of stress granules and from aggresomes.
Curiously, vaultosomes were induced by treatment of cells with ALLN, which
is an inhibitor of neutral cysteine proteases. This result suggests that
thiol-modified proteins may accumulate in the vicinity of vaultosomes and that
vaults may be involved in the complex biochemical sensing and response to
perturbations in the thiol:redox buffering system. It has been known for quite
some time that selenium and tellurium compounds are thiol reactive reagents
and all of these oxyanions perturb the thiol:redox buffering system
(Bersin and Logemann, 1933;
Valko et al., 2006). Our study
shows that vaults respond dynamically to these toxic oxyanions. The large size
of the vault interior indicates that vaults could harbor macromolecular
complexes at the cell surface in response to tellurite uptake. Recent work
shows that the vault shell can open and close transiently
(Poderycki et al., 2006).
Whether vaults contain complexes of organic telluroproteins
(Ramadan et al., 1989),
macromolecular complexes involved in the reduction of tellurite to elemental
tellurium (Deuticke et al.,
1992; Turner et al.,
1992), or components of the thiol:redox system remains to be
determined.
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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