The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1–871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1–RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1–RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1–RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding

CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

ABSTRACT We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.</jats:p

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding

Partially purified murine TEP1–RBD interacts with TR but has limited specificity

<p><b>Copyright information:</b></p><p>Taken from "The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle"</p><p>Nucleic Acids Research 2005;33(3):893-902.</p><p>Published online 8 Feb 2005</p><p>PMCID:PMC549401.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Amino acids 201–871 of murine TEP1 were expressed in and partially purified using the hexahistidine tag derived from the pET28a vector. Shown are a Coomassie-stained gel (left panel) and western blot (right panel) using anti-TEP1 polyclonal antibodies. () EMSA of nt 1–223 of mouse TR incubated with TEP1–RBD. Five fmol P-labeled probe was incubated with 0, 5, 20 and 80 ng TEP1–RBD (lanes 1–4). Probe was competed off with 50× full-length TR (lane 5) and increasing amounts of mVR double point mutant (lanes 7 and 8), but not wild-type mVR (lane 6). The 250× unlabeled 5S Ribosomal RNA, tRNA or an artificial RNA (NL15) do not compete with the probe (lanes 7–9), but 50× excess of an artificial RNA derived from the pBluescript polylinker region does compete efficiently (lane 10). Labeled RNA is indicated with a black dot and shifted complexes are indicated with an asterisk. () Immunoprecipitation of TEP1–RBD from binding reactions using anti-T7 monoclonal antibody co-immunoprecipitates the TR transcript. Equivalent amounts of the bound (B) and unbound (U) fraction were analyzed by either western blot using anti-TEP1 polyclonal antibody (upper panel) or by fractionation on a 10% acrylamide/8 M urea gel (lower panel). The latter gel was dried and radioactive bands visualized by phosphorimager analysis. Antibodies to the T7 epitope (lanes 1 and 2), but not antibodies to the FLAG (lanes 3 and 4) and VSVG (lanes 5 and 6) epitopes, immunoprecipitated both TEP1–RBD and TR

Targeting Vault Nanoparticles to Specific Cell Surface Receptors

As a naturally occurring nanocapsule abundantly expressed in nearly all-eukaryotic cells, the barrel-shaped vault particle is perhaps an ideal structure to engineer for targeting to specific cell types. Recombinant vault particles self-assemble from 96 copies of the major vault protein (MVP), have dimensions of 72.5 × 41 nm, and have a hollow interior large enough to encapsulate hundreds of proteins. In this study, three different tags were engineered onto the C-terminus of MVP: an 11 amino acid epitope tag, a 33 amino acid IgG-binding peptide, and the 55 amino acid epidermal growth factor (EGF). These modified vaults were produced using a baculovirus expression system. Our studies demonstrate that recombinant vaults assembled from MVPs containing C-terminal peptide extensions display these tags at the top and bottom of the vault on the outside of the particle and can be used to specifically bind the modified vaults to epithelial cancer cells (A431) via the epidermal growth factor receptor (EGFR), either directly (EGF modified vaults) or as mediated by a monoclonal antibody (anti-EGFR) bound to recombinant vaults containing the IgG-binding peptide. The ability to target vaults to specific cells represents an essential advance toward using recombinant vaults as delivery vehicles