Multi-subunit protein complexes control membrane fusion events in eukaryotic cells (1). CORVET and HOPS are two such multi-subunit complexes, both containing the Sec1/Munc18 protein subunit VPS33A (2). Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. Recent research suggests that VPS33B and VIPAR comprise two subunits of a novel multi-subunit complex analogous in configuration to CORVET and HOPS (3).
In a recent publication (4), Hunter and colleagues, further characterized the VPS33B and VIPAR complex. Using co-immunoprecipitation and proximity-based ligation assay, they identified two novel VPS33B-interacting proteins, VPS53 and CCDC22.
In vitro binding experiments, VPS33B and GST-VIPAR were co-expressed in Escherichia coli and purified by GSH affinity. The VPS33B/GSTVIPAR complex was used as bait in pulldown experiments, with myc-CCDC22 and myc-VPS53 expressed by cell-free in vitro transcription/translation in wheat germ lysate. Myc-CCDC22 was very efficiently pulled down by VPS33B/GST-VIPAR, whereas myc-VPS53 was not .The interaction between VPS53 and the VPS33B-VIPAR complex was either indirect, requires other proteins contribute to the interaction, or requires a post-translational modification not conferred in the plant cell-free expression system (wheat germ). Pull-down experiments with individual subunits or expressing as complexes, was inefficient and did not result in binding to VPS33B/GST-VIPAR.
To further understand how VPS33B-VIPAR may interact with CCDC22, Hunter and colleagues attempted to refine the region of CCDC22 that interacts with VPS33B/GST-VIPAR by generating a series of truncated forms of CCDC22. However, none of five CCDC22 truncations were able to bind to VPS33B/GST-VIPAR. The hypothesis was that truncated forms of CCDC22 are unstable and unable to fold correctly in this assay system.
Additional experiments noted that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit protein complex.
- D’Agostino, M. et. al. (2017) A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638.
- Balderhaar, H. J. K. and Ungermann, C. (2013) CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. J. Cell Sci. 126, 1307–16.
- Spang, A. (2016) Membrane Tethering Complexes in the Endosomal System. Front. Cell Dev. Biol. 4, 35.
- Hunter, M. et. al. (2017) Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B. [Internet bioRxiv http://dx.doi.org/10.1101/236695 Accessed 3/12/2018]
ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;
Transcriptional activator-like effector nucleases (TALENs) have rapidly become a technique of choice for precision genome engineering. TALENs are custom-designed nucleases that consist of a modular DNA-binding domain fused to a monomeric, C-terminal FokI nuclease domain (1). TALENs work in pairs and are designed to recognize and bind to tandem-oriented sequences in genomic DNA, separated by a short spacer (15–30 bp). TALEN binding causes dimerization and activation of the FokI nuclease domains, which results in cleavage of the DNA within the spacer region. Small insertions or deletions (indels) are frequently introduced at this site, as the result of errors made during DNA repair by nonhomologous end-joining (NHEJ). These indels can be up to several hundred base pairs in length and result in frameshift mutations that lead to the production of truncated or nonfunctional proteins.
Successful use of TALENs for inducing targeted mutations has been reported in many conventional models, for example: mice, Xenopus and D. melanogaster. TALENs are also reported to be functional in a variety of other invertebrate arthropods, including mosquitos,silkworm and cricket. A recent publication (2) illustrates the use of TALEN technology for the genetic manipulation in P. dumerilii (marine ragworm). Continue reading
Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in traditional E. coli expression systems.
In his webinar, “In vitro, cell-free protein expression–How it helps speed up your research”, Gary Kobs offered an overview of the different cell-free expressions systems offered by Promega and highlighted what needs the different systems best address. He discussed different applications of cell-free expressed proteins and highlighted the combined uses of the HaloTag® Technology with cell-free protein expression. Continue reading
Protein phosphorylation is one of the most biologically relevant modifications and is involved in many eukaryotic and prokaryotic cellular signaling processes. It is estimated that one-third of human proteins are phosphorylated.
The following examples utilize the ability of cell free experession to express active proteins, and when supplemented with the necessary components (e.g., ATP, NaCl), to be used for the characterization of phosphorylation events.
Modrof, J. et al. (2005) Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies. J. Vir. 79, 10023–31. Use of TNT® cell-free to express NS2 and NS2 mutant proteins for use in vitro kinase assays to confirm phosphorylation by protein kinase CK2.
Kwon, S. et al. (2005) Signal pathway of hypoxia-inducible factor-1alpha phosphorylation and its interaction with von Hippel-Lindau tumor suppressor protein during ischemia in MiaPaCa-2 pancreatic cancer cells. Clin. Cancer Res. 11, 7607–13. The TNT® system was used to identify which p38 mitogen-activated protein kinase isoform(s) was cabable of phosphorylation of HIF—1 alpha
Harris, J. et al. (2006). Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon. J. Immunol. 177, 2527–35. IKK and IKK mutants were expressed using TNT and used in a vitro kinase assay to characterize the recognition motif in cRel transcription domain
Jailais, Y. et al. (2011) Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–37. Using a vitro kinase assay, full–length and truncations versions of the Brassinostediod-insentive receptor protein were expressed using the TNT® system and incubated with purified BR11 kinase domain to determine binding sites of the two proteins.
Cell free protein expression can be utilized for the analysis of: protein/protein interactions, protein nucleic acid interactions, analysis of post translational modifications and many other applications. The majority of these references are based on the characterization of mammalian proteins.
However there are several references using TNT-based systems (either rabbit reticulocyte lysate or wheat germ based) for the analysis of proteins from plants, examples include: Continue reading