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Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis

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摘要

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3- independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects.

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  • 来源
    《Molecular and Cellular Biology》 |1995年第3期|1210-1219|共10页
  • 作者

    Zachary W. Pitluk; Michele Mcdonough; Pitchai SanganDavid K. Gonda;

  • 作者单位

    Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024;

    Department of Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook, NY 11794;

    Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, 333 Ceder St., P. O. Box 208024, SHM C-34, New Haven, CT 06520-8024;

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  • 原文格式 PDF
  • 正文语种 英语
  • 中图分类 分子生物学;
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