Abstract
The stabilities of many key proteins are regulated, e.g. via ubiquitination and proteasomal degradation, with important biological consequences. We present a convenient method that allows the analysis and comparison of protein stabilities during embryogenesis using early zebrafish development as a model system. Basically, this method involves ectopic overexpression of epitope-tagged proteins via mRNA injections in one-to-four-cell stage embryos and subsequent protein detection after various time points. Indeed, the protein stability of the ubiquitin ligase RLIM, which is able to autoubiquitinate and target itself for proteasomal degradation, was much shorter when compared to a protein consisting of a Myc epitope-tag and a nuclear localization domain. Thus, this method may be used more widely for the study of developmental protein stability.
Similar content being viewed by others
References
Pickart CM (2001). Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503–533.
Bach I, Ostendorff HP (2003). Orchestrating nuclear functions: Ubiquitin sets the rhythm. Trends Biochem Sci 28(4): 189–195.
Freiman RN, Tjian R (2003). Regulating the regulators. Lysine modifications make their mark. Cell 112(1): 11–17.
Hicke L (2001). A new ticket for entry into budding vesicles-ubiquitin. Cell 106(5): 527–530.
Kloetzel PM (2001). Antigen processing by the proteasome. Nat Rev Mol Cell Biol 2(3): 179–187.
Bach I (2000). The LIM domain: Regulation by association. Mech Dev 91(1- 2): 5–17.
Hobert O, Westphal H (2000). Functions of LIM-homeobox genes. Trends Genet 16(2): 75–83.
Ostendorff HP, Peirano RI, Peters MA, Schlüter A, Bossenz M, Scheffner M et al. (2002). Ubiquitination-dependent cofactor exchange on LIM homeodomain transcription factors. Nature 416(6876): 99–103.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995). Stages of embryonic development of the zebrafish. Dev Dyn 203(3): 253–310.
Schultheiss TM, Xydas S, Lassar AB (1995). Induction of avian cardiac myogenesis by anterior endoderm. Development 121(12): 4203–4214.
Becker T, Ostendorff HP, Bossenz M, Schlüter A, Becker CG, Peirano RI et al. (2002). Multiple functions of LIM domain-binding CLIM/NLI/Ldb cofactors during zebrafish development. Mech Dev 117(1- 2): 75–85.
Becker CG, Schweitzer J, Feldner J, Becker T, Schachner M (2003). Tenascin-R as a repellent guidance molecule for developing optic axons in zebrafish. J Neurosci 23(15): 6232–6237.
Bach I, Rodriguez-Esteban C, Carriere C, Bhushan A, Krones A, Rose DW et al. (1999). RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex. Nat Genet 22(4): 394–399.
Roth MB, Zahler AM, Stolk JA (1991). A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol 115(3): 587–596.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Becker, T., Bossenz, M., Tursun, B. et al. Comparing protein stabilities during zebrafish embryogenesis. Methods Cell Sci 25, 85–89 (2003). https://doi.org/10.1023/B:MICS.0000006895.03556.9b
Issue Date:
DOI: https://doi.org/10.1023/B:MICS.0000006895.03556.9b