Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport,
transcriptional regulation, apoptosis, protein stability and response to stress.
In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.
Cell free expression can be used to characterize sumoylation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with necessary addition components,). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels, when compared to control reactions.
The following references illustrate the use of cell free expression for this application.
Brandl, A. et al. (2012) Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress. J. Mol. Cell. Biol. (online only)
Janer, A. et al. (2010). SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7. Human. Mol. Gen. 19, 181—95.
Rytinki, M. et al. (2009) SUMOylation attenuates the function of PGC-1alpha. J. Biol. Chem. 284, 26184-93.
Klein, U. et al. (2009) RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin. Mol. Cell. Biol. 20, 410–18.
Seo, W. and Ziltener, H. (2009) CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis. Blood, 114, 3567–77.
Protein location: outer mitochondrial membrane (Yeast in vitro import assay)
Curado, S. et.al. (2010) Dis.Mod. Mech. 3, 486-95. PubMed ID 20483998.
Chemically mutagenized zebra fish were assayed for liver defects in their F3 progeny.This screen led to the identification of mutant called oliver. Oliver mutants have an o-shaped liver of a much deprived size due to the depletion of most of the hepatocytes. This mutation maps to the Tomm22gene which encodes a translocase of the outer membrane and thought to play an important role in protein import into mitochondria. Various Tomm22 mutants were expressed and used in a yeast in vitro import systemto determine if correct inserted into the yeast outer mitochondrial membrane.
Protein modification: hydroxylation
Serchov, T. et.al. (2010) J. Biol. Chem. 285, 21223-232. PubMed ID 20418372 .
Proline hydroxylation is also a vital component of hypoxia via hyposxia inducible factors. The cellular response to hypoxia involves the induction of the hypoxia-inducible factor considered to be the major transcription factor involved in gene regulation of hypoxia. This factor is hydroxylated by prolyl-hydroxase dolman proteins (PHDs). To investigate if a newly identified component of the hypoxia pathway (Elk3) is also hydroxylated, proteins were expressed +/- PHDs cofactors and protein mobility was measured via gel analysis.
Gene Experession: Programmed Ribosomal Frameshift
Kobayashi, Y. et.al. (2010) J. Biol. Chem. 285, 19776-784. PubMed ID 20427288.
Programmed -1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses (HIV-1 for example). Recently a genome-wide screen demonstrated that down regulation of eukaryotic release factor (eRF1) inhibited HIV-1 replication. In order to characterize the dose dependent effect of eRF1, increasing amounts were expressed in the presence of dual luciferase reporter vectors harboring a HIV-1 PRF signal