Dynein light chain LC8 promotes assembly of the coiled-coil domain of swallow protein

Biochemistry. 2004 Apr 20;43(15):4611-20. doi: 10.1021/bi036328x.

Abstract

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that is thought to play a fundamental role in both the assembly of the motor complex and the recruitment of cargo. An interaction between LC8 and the Drosophila protein swallow has been previously characterized and supports a role for dynein in the localization of maternal morphogens during oogenesis. Swallow is required for the proper localization of bicoid mRNA, the anterior determinant that plays a critical role in establishment of the Drosophila embryonic axis. In this work, we prepared constructs of swallow, each containing a predicted coiled-coil domain and variable surrounding segments that lie within the domain proposed to interact with LC8. The interaction between LC8 and swallow domains was characterized by glutathione S-transferase (GST) pull-down assays, limited proteolysis followed by mass spectrometry, and circular dichroic spectroscopy. Hydrodynamic measurements, covalent cross-linking, and circular dichroic spectroscopy show that this domain of swallow is an unstable dimeric coiled-coil. Upon LC8 binding, however, the coiled-coil becomes significantly more stable. A possible general role for LC8 in macromolecular assembly is discussed.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Circular Dichroism
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Dyneins
  • Genetic Vectors
  • Glutathione Transferase / genetics
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Protein Binding / genetics
  • Protein Folding
  • Protein Interaction Mapping
  • Protein Processing, Post-Translational* / genetics
  • Protein Structure, Tertiary / genetics
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / chemical synthesis
  • Recombinant Fusion Proteins / metabolism
  • Thermodynamics

Substances

  • Carrier Proteins
  • Drosophila Proteins
  • Peptide Fragments
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • ctp protein, Drosophila
  • swa protein, Drosophila
  • Glutathione Transferase
  • Dyneins