The E3 ligase CUL3/RDX controls centromere maintenance by ubiquitylating and stabilizing CENP-A in a CAL1-dependent manner

Dev Cell. 2014 Mar 10;28(5):508-19. doi: 10.1016/j.devcel.2014.01.031.

Abstract

Centromeres are defined by the presence of the histone H3 variant CENP-A in a subset of centromeric nucleosomes. CENP-A deposition to centromeres depends on a specialized loading factor from yeast to humans that is called CAL1 in Drosophila. Here, we show that CAL1 directly interacts with RDX, an adaptor for CUL3-mediated ubiquitylation. However, CAL1 is not a substrate of the CUL3/RDX ligase but functions as an additional substrate-specifying factor for the CUL3/RDX-mediated ubiquitylation of CENP-A. Remarkably, ubiquitylation of CENP-A by CUL3/RDX does not trigger its degradation but stabilizes CENP-A and CAL1. Loss of RDX leads to a rapid degradation of CAL1 and CENP-A and to massive chromosome segregation defects during development. Essentially, we identified a proteolysis-independent role of ubiquitin conjugation in centromere regulation that is essential for the maintenance of the centromere-defining protein CENP-A and its loading factor CAL1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Centromere / physiology*
  • Centromere Protein A
  • Cullin Proteins / physiology*
  • DNA-Binding Proteins / metabolism*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila Proteins / physiology*
  • Drosophila melanogaster / embryology
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism*
  • Female
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Regulation, Developmental
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Histones / metabolism*
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins / physiology*
  • Male
  • Mutagenesis, Site-Directed
  • Mutation
  • Proteolysis
  • RNA, Small Interfering / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Two-Hybrid System Techniques
  • Ubiquitin / metabolism*
  • Ubiquitination

Substances

  • Cal1 protein, Drosophila
  • Centromere Protein A
  • Cid protein, Drosophila
  • Cul3 protein, Drosophila
  • Cullin Proteins
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • RNA, Small Interfering
  • Rdx protein, Drosophila
  • Recombinant Fusion Proteins
  • Ubiquitin
  • Green Fluorescent Proteins