Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.
We would like to thank Janice Fischer, Fen-Biao Gao, Zoltán Lipinszki, Bloomington Stock Center, the DRSC, and The Developmental Studies Hybridoma Bank-DSHB (University of Iowa) for flies, cells, dsRNA templates, and antibodies, and David Gubb for helpful advice and support. We thank J. D. Sutherland for his suggestion to use the anti-GFP beads. We would also like to thank Larry Reiter and Catherine Lindon for critical reading and comments on the manuscript. We acknowledge the CIC bioGUNE Gene Silencing Platform for support. This work was supported by a Basque Government research grant (PI2011-24) and a March of Dimes Basil O′Connor Starter Scholar Research Award (5-FY12-16) to U.M. RB thanks the Spanish MICINN (grants BFU2008-01884, BFU2011-25986) and the Consolider Program (CSD2007-008-25120), the Basque Government (PI2009-16 and PI2012/42), and the Bizkaia County.
Conflict of interest
The authors declare that they have no conflicts of interest.
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