The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation.
The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation. In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates. The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution. Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human. We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein. The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates. In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes. The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect. The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis. E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.
Watkins
J F
JF
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Sung
P
P
Prakash
S
S
Prakash
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D004260:DNA Repair / Q000235:genetics*; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes
D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases