Abstract
Protein lysine acetyltransferases (KATs) catalyze acetylation of the ε-amino group on a specific lysine residue, and this posttranslational modification is important for regulating the function and activities of thousands of proteins in diverse organisms from bacteria to humans. Interestingly, many known KATs exist in multisubunit complexes and complex formation is important for their proper structure, function, and regulation. Thus, it is necessary to reconstitute enzymatically active complexes for studying the relationship between subunits and determining structures of the complexes. Due to inherent limitations of bacterial and mammalian expression systems, baculovirus-mediated protein expression in insect cells has proven useful for assembling such multisubunit complexes. Related to this, we have adopted such an approach for reconstituting active tetrameric complexes of monocytic leukemia zinc (MOZ, finger protein, recently renamed MYST3 or KAT6A) and MOZ-related factor (MORF, also known as MYST4 or KAT6B), two KATs directly linked to development of leukemia and self-renewal of stem cells. Herein, we use these complexes as examples to describe the related procedures. Similar methods have been used for reconstituting active complexes of histone deacetylases, lysine demethylases, and ubiquitin ligases, so this simple approach can be adapted for molecular dissection of various multisubunit complexes.
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Acknowledgments
We acknowledge that the f-pAcSG2 plasmid was initially constructed when X.J.Y. was a postdoctoral fellow in Dr. Yoshihiro Nakatani’s laboratory at National Institutes of Health (NIH), the USA. This work was supported by operating grants from Canadian Cancer Society and Canadian Institutes of Health Research (to X.J.Y.).
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Yan, K., Wu, CJ., Pelletier, N., Yang, XJ. (2012). Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_29
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DOI: https://doi.org/10.1007/978-1-61779-376-9_29
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