The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain

EMBO J. 1999 Dec 1;18(23):6832-44. doi: 10.1093/emboj/18.23.6832.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Fungal Proteins / chemistry*
  • Glutathione Transferase / metabolism
  • Ligases / chemistry*
  • Molecular Sequence Data
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Two-Hybrid System Techniques
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases*
  • Ubiquitins / chemistry*

Substances

  • Fungal Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitins
  • Ubiquitin-Conjugating Enzymes
  • UBR1 protein, S cerevisiae
  • Ubiquitin-Protein Ligases
  • Glutathione Transferase
  • Ligases