CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins.

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.

A minor catalytic activity of Src family kinases is sufficient for maximal activation of mast cells via the high-affinity IgE receptor.

Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcepsilonRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn(-/-) bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn(-/-) cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn(-/-) cells. Restoration of wild-type and WeeB lyn alleles in lyn(-/-) cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector-transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.

Generation of iPS cells using a BacMam multigene expression system.

Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64-98%. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15-24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.

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.

Identification of conserved vault RNA expression elements and a non-expressed mouse vault RNA gene.

Vault RNA (vRNA) genes have been cloned from several vertebrates including rat, mouse, and humans. Their copy numbers vary, as does the length of the encoded RNA. We have determined that the mouse genome contains two vRNA genes; one is expressed the other is a pseudogene. In vitro transcription of the rat vRNA gene by RNA polymerase III has previously been shown to be dependent on a combination of both external and internal promoter sequence elements. By comparing the upstream regions of the vertebrate vRNA genes, a 25 bp conserved sequence and a TATA box can be identified. Furthermore, the unique arrangement of the internal promoter boxes (one A and two B boxes) is conserved in the expressed human vRNA genes even though a new RNA polymerase III termination sequence has evolved between the two B boxes.

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 x 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.

The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior.

Vaults are 13 million Da ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may functionally interact with the cellular milieu.

The La RNA-binding protein interacts with the vault RNA and is a vault-associated protein.

Vaults are highly conserved ubiquitous ribonucleoprotein particles with an undefined function. Three protein species (p240/TEP1, p193/VPARP, and p100/MVP) and a small RNA comprise the 13-MDa vault particle. The expression of the unique 100-kDa major vault protein is sufficient to form the basic vault structure. Previously, we have shown that stable association of the vault RNA with the vault particle is dependent on its interaction with the p240/TEP1 protein. To identify other proteins that interact with the vault RNA, we used a UV-cross-linking assay. We find that a portion of the vault RNA is complexed with the La autoantigen in a separate smaller ribonucleoprotein particle. La interacts with the vault RNA (both in vivo and in vitro) presumably through binding to 3'-uridylates. Moreover, we also demonstrate that the La autoantigen is the 50-kDa protein that we have previously reported as a protein that co-purifies with vaults.