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BMC Dev Biol
2005 Feb 14;5:3. doi: 10.1186/1471-213X-5-3.
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Sea urchin vault structure, composition, and differential localization during development.
Stewart PL
,
Makabi M
,
Lang J
,
Dickey-Sims C
,
Robertson AJ
,
Coffman JA
,
Suprenant KA
.
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BACKGROUND: Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis.
RESULTS: The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven approximately 50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14-55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis.
CONCLUSIONS: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.
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15710043
???displayArticle.pmcLink???PMC550661 ???displayArticle.link???BMC Dev Biol ???displayArticle.grants???[+]
Figure 1. The molecular composition of the sea urchin vault.(A) SDS-PAGE analysis (4–16% acrylamide gradient gel) of isolated sea urchin (S) and rat (R) vaults. The band at ~100 kDa in both lanes represents the major vault protein (MVP). (B) SDS-PAGE analysis of sea urchin vaults both without (-) and with (+) RNase treatment. The 26.5 kDa band in the (-) RNase lane, which is missing in the (+) RNase lane, is thought to correspond to the sea urchin vRNA. Mr represents the molecular weight marker lane.
Figure 2. The sequence of the sea urchin major vault protein (SpMVP). (A) Amino acid sequence of SpMVP aligned with that of rat MVP. Identical residues are highlighted in yellow, and similar residues are highlighted in green. (B) Bar diagram of SpMVP. The sequence has seven repeats in the N-terminal half of the protein (R1-R7), each repeat consisting of 41 to 62 aa, spanning residues 32–400; and a predicted coiled coil region in the C-terminal half of the sequence (C.C.), residues 663–762. The positions of two possible nuclear localization sequences (NLS1 470-KKAR, and NLS2 498-KPKR), a putative nuclear export sequence (NES, 792–816), and two probable sumoylation sites that are conserved across six species (S1 K308-VKGE, and S2 K707-AKAE) are indicated.
Figure 3. Western blots of isolated sea urchin vaults. (A) Peptide amino acid sequence of rat MVP (aa 19–35) aligned with Sp MVP (25–41). Anti-peptide antibodies were generated against the rat MVP sequence shown and affinity-purified as previously described [38]. Twelve of the rat MVP amino acids are conserved in the Sp MVP peptide sequence. (B) 150 μg of sea urchin egg extract proteins (lane E) and 10 μg of purified sea urchin vault proteins (lane V) were separated on this Coomassie-blue stained SDS-8% polyacrylamide mini-gel. The arrow shows the position of the 100 kDa MVP in (B, C and D). (C) Alkaline-phosphatase stained western blot showing the migration of the pre-stained protein ladder (lane M: 176.5, 113.7, 80.9, 63.8 (pink), 49.5, and 8.4 kDa polypeptides). Affinity-purified anti-sea urchin MVP antibodies [16] recognize a 100 kDa polypeptide in egg extracts (lane E) and in purified vault preparations (lane V). Asterisks indicate two bands of approximately 80 kDa and 50 kDa that may be breakdown products of the MVP. (D) Affinity-purified anti-peptide antibodies (anti-LDQN, [38]) bind to the 100 kDa MVP polypeptide in purified sea urchin vaults. This peptide antibody appears to bind non-specifically to a large number of polypeptides in the sea urchin egg extracts.
Figure 4. Negative-stain and cryoEM images of isolated sea urchin vaults. (A) Negative-stain electron micrograph. (B) Cryoelectron micrograph. The black arrow indicates a vault that is opening at the midsection, and the white arrow indicates the dark molecular contents within another particle. The scale bar represents 1,000 Å. Note that the magnification of the cryoEM image (B) is slightly higher than that of the negative-stain EM image (A).
Figure 5. The sea urchin vault reconstruction at 33 Å resolution. (A) The full reconstruction, which reveals that the sea urchin vault has essentially the same exterior structure as rat and mouse vaults. (B and C) The reconstruction is shown cropped along two perpendicular axes to reveal the hollow interior. The crop planes are displayed with the strongest density in red and the weakest density in green. Note that the strongest density is in the "shoulder" region at the top and bottom of the central barrel section. The flat portion of one cap is indicated by an arrow in (B). The scale bar represents 100 Å.
Figure 6. Confocal images of sea urchin embryos at various developmental stages. The cellular distribution of MVP is revealed by staining with an affinity-purified polyclonal antiserum against the sea urchin MVP and secondary staining with Oregon Green-conjugated anti-rabbit antibody (A, D, G, J; left column). The nuclei are stained with DAPI (B, E, H, K; middle column). By merging the MVP and DNA images coincident staining is observed as cyan (C, F, I, L; right column). Note that as development proceeds the cellular localization of MVP progressively shifts from cytoplasmic to nuclear.
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