Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

Nat Commun. 2016 Jun 16:7:11874. doi: 10.1038/ncomms11874.

Abstract

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cattle
  • Centrioles / metabolism*
  • Centrioles / ultrastructure
  • Cilia / metabolism*
  • Cilia / ultrastructure
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins / chemistry*
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Swine
  • Tubulin / chemistry*
  • Tubulin / genetics
  • Tubulin / metabolism

Substances

  • CENPJ protein, human
  • Microtubule-Associated Proteins
  • Protein Isoforms
  • Recombinant Proteins
  • Tubulin