A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.
The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation 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 ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did 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. Inhibition 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-terminus 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 ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.
Breitschopf
K
K
Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa.
Bengal
E
E
Ziv
T
T
Admon
A
A
Ciechanover
A
A
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D008239:Lysine / Q000378:metabolism*; D017570:MyoD Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011849:Radioactive Tracers; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism* / Q000502:physiology
D017570:MyoD Protein; D010446:Peptide Fragments; D011849:Radioactive Tracers; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D008239:Lysine