The N-terminal domain of MyoD is necessary and sufficient for its nuclear localization-dependent degradation by the ubiquitin system.
A growing number of proteins, including the myogenic transcription factor MyoD, are targeted for proteasomal degradation after N-terminal ubiquitination (NTU) where the first ubiquitin moiety is conjugated to the N-terminal residue rather than to an internal lysine. NTU might be essential in targeting both lysine-containing and naturally occurring lysine-less proteins such as p16(INK4a) and p14(ARF); however, the mechanisms that underlie this process are largely unknown. Specifically, the recognition motif(s) in the target substrates and the ubiquitin ligase(s) that catalyze NTU are still obscure. Here we show that the N-terminal domain of MyoD is critical for its degradation and that its destabilizing effect depends on nuclear localization of the protein. Deletion of the first 15 aa of MyoD blocked completely its lysine-independent degradation. Importantly, transfer of the first 30 N-terminal residues of MyoD to GFP destabilized this otherwise stable protein, and, here too, targeting for degradation depended on localization of the protein to the nucleus. Deletion of the N-terminal domain of lysine-less MyoD did not abolish completely ubiquitination of the protein, suggesting that this domain may be required for targeting the protein also in a postubiquitination step. Interestingly, NTU is evolutionarily conserved: in the yeast Saccharomyces cerevisiae lysine-less (LL) MyoD is degraded in a ubiquitin-, N-terminal domain-, and nuclear localization-dependent manner. Taken together, our data suggest that a short N-terminal segment of MyoD is necessary and sufficient to render MyoD susceptible for ubiquitin- and nuclear-dependent degradation.
Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Ronen
R
Sadeh
Kristin
K
Breitschopf
Beatrice
B
Bercovich
Muhammad
M
Zoabi
Yelena
Y
Kravtsova-Ivantsiv
Daniel
D
Kornitzer
Alan
A
Schwartz
Aaron
A
Ciechanover
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D008239:Lysine; D051379:Mice; D017570:MyoD Protein / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism*
D017570:MyoD Protein; D025801:Ubiquitin; D008239:Lysine