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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

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The major vault protein is related to the toxic anion resistance protein (TelA) family

Kathy A. Suprenant^1,*, Nathan Bloom¹, Jianwen Fang² and Gerald Lushington³

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

View larger version (40K): [in this window] [in a new window]   Fig. 1. Sequence similarity between the mammalian major vault protein (MVP) and the bacterial tellurite resistance protein (TelA). (A) Schematic diagram of the major vault protein (MVP). Human MVP is composed of an amino-terminal domain (aa 26–401) comprising a series of seven imperfect repeats (blue circles) and a carboxyl-terminal region (black bar) that contains a coiled-coil domain (aa 652–800). (B) Sequence alignment of the repeat unit of MVP with TelA. Residue number corresponds to the consensus gapped sequence and thus does not correspond perfectly to that of either constituent sequence. Amino acid symbols are colored according to quality of match, with red indicating perfect conservation (redundantly labeled with a asterisk in the bottom line), green indicating substantial similarity (colon, strong similarity; stop, moderate similarity) and light blue indicating a mismatch. The line labeled `Prim. Cons.' reports known residues within the consensus sequence (dark blue script). The portion of the MVP sequence structurally resolved in NMR studies (Kozlov et al., 2005) is underlined with a bar colored according to observed secondary structure: magenta for helices, yellow for beta strands, and black for coils and turns.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Relative structures of our model for the TelA protein and its associated MVP template, shown as (A) overlapping ribbon diagrams and (B) juxtaposed solvent accessible surfaces for TelA (left) and MVP (right). Ribbon coloring of MVP in A corresponds to observed secondary structure elements: magenta=helices, yellow=beta strands/sheets, cyan=coils/turns, while for contrast the TelA backbone is rendered as a monochromatic blue tube. Surface rendering in B reflects relative electrostatics, with the following color scheme: N,H portions of cationic side chains are blue, anionic carbonyl oxygens are red, other neutral H-bond acceptor atoms are magenta, donatable protons are cyan, polar aliphatic groups are white and all hydrophobic groups are yellow.

View larger version (61K): [in this window] [in a new window]   Fig. 3. MVP and VPARP accumulations in the presence of tellurite. (A) Fluorescence micrograph of an untreated HeLa cell stained with an antibody against the major vault protein (LDQN) (red) and counterstained with DAPI. Bar, 10 µm. (B) Pronounced MVP aggregates (arrows) are revealed when HeLa cells are grown at 37°C for 30 min in the presence of 0.5 mmol l–1 K2TeO3. (C) The formation of MVP aggregates is temperature dependent. HeLa cells were cultured at 37°C and then incubated in the presence (filled bars) and absence (open bars) of 0.5 mmol l–1 K2TeO3 for 30 min at the temperatures shown. The number of cells with MVP aggregates (approximately 2 µm or larger) were counted and expressed as a percentage of the total cells. At least 100 cells were examined at each temperature and the experiment was done in triplicate. Values are reported as mean % ± s.d. (D) The percentage of cells with MVP aggregates increases over a 1 h incubation. HeLa cells were incubated in the presence of 0.5 mmol l–1 K2TeO3 for 30 min and the percent of cells with aggregates were quantified as described above. (E) Nearly 20% of the HeLa cells incubated in 5 mmol l–1 K2TeO3 will form MVP aggregates. The higher the concentration of K2TeO3, the greater the percentage of HeLa cells with MVP aggregates. (F) Fluorescence micrograph of untreated HeLa cells stained with an antibody against the vault-associated poly(ADP-ribosyl)polymerase (VPARP) and counterstained with DAPI. Bar, 10 µm. (G) VPARP accumulations also appear at the cell periphery during a 30 min incubation in the presence of 0.5 mmol l–1 K2TeO3.

View larger version (82K): [in this window] [in a new window]   Fig. 4. Vault aggregation in normal normal human vascular endothelial cells (HUVECs) and the HeLa cancer cell line. HUVECs were incubated at 37°C for 30 min in the presence (A) or absence (B) of 0.5 mmol l–1 K2TeO3 and subsequently stained with antibodies against MVP (red) and tubulin (green). MVP aggregates form at the cell periphery in HUVECs (arrows). In addition, HeLa cells were incubated at 37°C for 30 min in the presence or absence of 0.5 mmol l–1 K2TeO3 and subsequently stained with tubulin and MVP antibodies (C–G). HeLa cells were subsequently washed three times in fresh medium without tellurite and incubated for an additional 24 h, fixed and processed for immunofluorescence. Accumulation of MVP at the cell periphery of HeLa cells is reversible. The MVP aggregates at the cell periphery (arrows, D) in the tellurite-treated cells are absent after a 24-h recovery period (E). In addition, the cell number has doubled in both the treated and untreated cultures. Occasionally, brightly-staining puncta form in response to tellurite treatment (asterisks). These bright MVP puncta are different from the MVP aggregates at the cell periphery in that these puncta stain with both MVP and tubulin. Bar, 20 µm.

View larger version (7K): [in this window] [in a new window]   Fig. 5. Bar graph showing the percentage of cells with vaultosomes following a 15 min treatment with 0.5 mmol l–1 arsenate, arsenite, selenate, selenite, vanadate and tellurite. Experiments were done in triplicate; values are means ± s.d.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Mammalian stress granules and vaultosomes do not colocalize. HeLa cells were incubated with 0.5 mmol l–1 sodium arsenite for 15 min at 37°C (D–F) or left untreated (A–C). Fluorescence micrographs show HeLa cells stained with antibodies against the RNA-binding protein TIA-1 (A,D) or MVP (B,E). Three-color overlays are shown in C and F. Vaultosomes (arrowhead in D) do not colocalize with TIA-1 proteins. Bar, 20 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Aggresomes and vaultosomes are distinct cytosolic entities. HeLa cells were incubated in the presence (D–F) and absence (A–C) of 10 mg ml–1 ALLN for 12 h and then processed for immunofluorescence with the FK2 antibodies against ubiquinated proteins (Ub) (A,D) or antibodies against MVP (B,E). Three color overlays are shown in C and F.