A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): Implications for the structure, function and evolution of the E2s

Elizabeth T. Silver, Todd J. Gwozd, Christopher Ptak, Mark Goebl, Michael J. Ellison

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains co-exist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis. Our observation that chimeric E2s can be constructed artificially that acquire new functions without loss of original functions, suggests that naturally occurring E2s are candidates for rapid evolutionary change. Finally, other evidence suggests a function for tails that has not been previously reported. Similar findings to those described here are reported in an accompanying manuscript (Kolman et al., 1992).

Original languageEnglish
Pages (from-to)3091-3098
Number of pages8
JournalEMBO Journal
Volume11
Issue number8
StatePublished - 1992

Fingerprint

Ubiquitin-Conjugating Enzymes
DNA Repair
Catalytic Domain
Cell Cycle
Repair
Cells
DNA
Peptides
Saccharomyces cerevisiae Proteins
Aptitude
Manuscripts
Ubiquitin
Individuality
Temperature
Derivatives
Enzymes
Growth
Chemical analysis

Keywords

  • CDC34 (UBC3)
  • Cell cycle
  • RAD6 (UBC2)
  • Ubiquitin conjugation
  • Yeast

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2) : Implications for the structure, function and evolution of the E2s. / Silver, Elizabeth T.; Gwozd, Todd J.; Ptak, Christopher; Goebl, Mark; Ellison, Michael J.

In: EMBO Journal, Vol. 11, No. 8, 1992, p. 3091-3098.

Research output: Contribution to journalArticle

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abstract = "The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains co-exist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis. Our observation that chimeric E2s can be constructed artificially that acquire new functions without loss of original functions, suggests that naturally occurring E2s are candidates for rapid evolutionary change. Finally, other evidence suggests a function for tails that has not been previously reported. Similar findings to those described here are reported in an accompanying manuscript (Kolman et al., 1992).",
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N2 - The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains co-exist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis. Our observation that chimeric E2s can be constructed artificially that acquire new functions without loss of original functions, suggests that naturally occurring E2s are candidates for rapid evolutionary change. Finally, other evidence suggests a function for tails that has not been previously reported. Similar findings to those described here are reported in an accompanying manuscript (Kolman et al., 1992).

AB - The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains co-exist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis. Our observation that chimeric E2s can be constructed artificially that acquire new functions without loss of original functions, suggests that naturally occurring E2s are candidates for rapid evolutionary change. Finally, other evidence suggests a function for tails that has not been previously reported. Similar findings to those described here are reported in an accompanying manuscript (Kolman et al., 1992).

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