A NOVEL SITE FOR UBIQUITINATION - THE N-TERMINAL RESIDUE, AND NOT INTERNAL LYSINES OF MYOD, IS ESSENTIAL FOR CONJUGATION AND DEGRADATION OFTHE PROTEIN

The ubiquitin proteolytic pathway is a major system for selective prot ein degradation in eukaryotic cells. One of the first steps in the deg radation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination, To date, this amino group has been the only known target for ubiquitin ation. Here we report that the N-terminal residue of MyoD is sufficien t and necessary for promotion of conjugation and subsequent degradatio n of the protein. Substitution of all lysine residues in the protein d id not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibiti on is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD, In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-termi nus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitinat ion are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocki ng of the alpha-NH2 group of wildtype protein renders it stable, while modification of the internal Lys residues with preservation of the fr ee N-terminal group left the protein susceptible to degradation, Our d ata suggest that conjugation of MyoD occurs via a novel modification i nvolving attachment of ubiquitin to the N-terminal residue. The polyub iquitin chain is then synthesized on an internal Lys residue of the li nearly attached first ubiquitin moiety.