Abstract
The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein-its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian EZ14k in E3 dependent ubiquitin - protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S.cerevisiae E230k, product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.
| Original language | English |
|---|---|
| Pages (from-to) | 2187-2193 |
| Number of pages | 7 |
| Journal | EMBO Journal |
| Volume | 10 |
| Issue number | 8 |
| State | Published - 1991 |
| Externally published | Yes |
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Keywords
- DNA repair
- E3
- Proteolysis
- RAD6
- Sporulation
ASJC Scopus subject areas
- Cell Biology
- Genetics
Cite this
Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme. / Sung, Patrick; Berleth, Erica; Pickart, Cecile; Prakash, Satya; Prakash, Louise.
In: EMBO Journal, Vol. 10, No. 8, 1991, p. 2187-2193.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme
AU - Sung, Patrick
AU - Berleth, Erica
AU - Pickart, Cecile
AU - Prakash, Satya
AU - Prakash, Louise
PY - 1991
Y1 - 1991
N2 - The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein-its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian EZ14k in E3 dependent ubiquitin - protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S.cerevisiae E230k, product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.
AB - The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein-its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian EZ14k in E3 dependent ubiquitin - protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S.cerevisiae E230k, product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.
KW - DNA repair
KW - E3
KW - Proteolysis
KW - RAD6
KW - Sporulation
UR - http://www.scopus.com/inward/record.url?scp=0025900036&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025900036&partnerID=8YFLogxK
M3 - Article
C2 - 2065660
AN - SCOPUS:0025900036
VL - 10
SP - 2187
EP - 2193
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 8
ER -