Identification of anaphase promoting complex substrates in S. cerevisiae

PLoS One. 2012;7(9):e45895. doi: 10.1371/journal.pone.0045895. Epub 2012 Sep 26.

Abstract

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential ubiquitin ligase that targets numerous proteins for proteasome-mediated degradation in mitosis and G1. To gain further insight into cellular pathways controlled by APC/C(Cdh1), we developed two complementary approaches to identify additional APC/C(Cdh1) substrates in budding yeast. First, we analyzed the stabilities of proteins that were expressed at the same time in the cell cycle as known APC/C substrates. Second, we screened for proteins capable of interacting with the Cdh1 substrate-binding protein in a yeast two-hybrid system. Here we characterize five potential APC/C substrates identified using these approaches: the transcription factors Tos4 and Pdr3; the mRNA processing factor Fir1; the spindle checkpoint protein kinase Mps1; and a protein of unknown function, Ybr138C. Analysis of the degradation motifs within these proteins revealed that the carboxyl-terminal KEN box and D-boxes of Tos4 are important for its interaction with Cdh1, whereas the N-terminal domain of Ybr138C is required for its instability. Functionally, we found that a stabilized form of Mps1 delayed cell division upon mild spindle disruption, and that elevated levels of Ybr138C reduced cell fitness. Interestingly, both Tos4 and Pdr3 have been implicated in the DNA damage response, whereas Mps1 regulates the spindle assembly checkpoint. Thus, the APC/C(Cdh1)-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaphase-Promoting Complex-Cyclosome
  • Cdh1 Proteins
  • Cell Cycle
  • Cell Proliferation
  • Gene Deletion
  • Gene Expression Regulation, Fungal*
  • Models, Biological
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Transcription, Genetic
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligase Complexes / chemistry
  • Ubiquitin-Protein Ligase Complexes / physiology*
  • mRNA Cleavage and Polyadenylation Factors / metabolism

Substances

  • CDH1 protein, S cerevisiae
  • Cdh1 Proteins
  • FIR1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • mRNA Cleavage and Polyadenylation Factors
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome
  • Protein-Serine-Threonine Kinases
  • MPS1 protein, S cerevisiae