The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain.
We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system.
Xie
Y
Y
Division of Biology, 147-75, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
Varshavsky
A
A
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D008025:Ligases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry*
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases