We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle development. Following binding to specific upstream DNA regulatory elements, it leads to activation of a wide array of muscle-specific genes and consequently to conversion of proliferating myoblasts to terminally differentiated mature myotubes (10,30,43). MyoD belongs to the family of muscle-specific basic helix-loop-helix (bHLH) proteins, which also includes Myf5, myogenin, and MRF4 (42,43). These myogenic regulators have 80% homology within a segment of about 70 amino acid residues that encompasses the basic and helix-loop-helix motifs. These motifs mediate DNA binding and dimerization, respectively (41). MyoD binds to DNA as a homodimer; however, a more stable complex is generated when MyoD heterodimerizes with other ubiquitously expressed bHLH proteins, such as E2A, E12, and E47 (28). The activity of MyoD is negatively regulated by members of the Id (inhibitors of differentiation) family of proteins. These proteins can heterodimerize with E12/E47 or MyoD, but since they lack the basic region, the complexes cannot bind to DNA and are therefore inactive (2, 24, 37).Like many other transcriptional factors, MyoD is an extremely short-lived protein, with a half-life of ϳ30 min (38). However, the proteolytic system(s) that targets the protein, as well as the ...

Most of the known cellular substrates of the ubiquitin system are short‐lived growth regulators and transcriptional activators. Very few enzymes involved in intermediary metabolism have been shown to be targeted by the system. In a reconstituted cell‐free system, we show that tyrosine aminotransferase (TAT), a key enzyme involved in amino acid metabolism, is conjugated and degraded in an ATP‐ and ubiquitin‐dependent manner. Degradation of ubiquitin–TAT adducts requires, in addition to the 26S proteasome, a novel, yet unidentified, factor. TAT can be protected from degradation by association with its coenzyme pyridoxal phosphate. To examine the potential role of the ubiquitin system in regulating the stability of the enzyme in vivo, we show that cell extracts derived from livers of animals in which TAT was induced, display a corollary increase in the formation of specific TAT–ubiquitin adducts. © 1997 Federation of European Biochemical Societies