The FLA3 KAP Subunit Is Required for Localization of kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

Mol Biol Cell. 2005 Mar;16(3):1341-54. doi: 10.1091/mbc.e04-10-0931. Epub 2004 Dec 22.

Abstract

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21 degrees C, but cannot maintain them at 33 degrees C. Although the Kinesin-2 complex is present at both 21 and 33 degrees C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Biological Transport
  • Blotting, Southern
  • Blotting, Western
  • Centromere / ultrastructure
  • Chlamydomonas / metabolism
  • Cilia / metabolism
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Epitopes / chemistry
  • Flagella / physiology*
  • Genetic Linkage
  • Kinesin / biosynthesis*
  • Kinesin / chemistry
  • Kinesin / metabolism*
  • Kinesin / physiology*
  • Microscopy, Fluorescence
  • Microscopy, Video
  • Models, Genetic
  • Molecular Sequence Data
  • Mutation
  • Phenotype
  • Protein Structure, Tertiary
  • RNA / chemistry
  • Sequence Homology, Amino Acid
  • Temperature
  • Time Factors

Substances

  • DNA, Complementary
  • Epitopes
  • RNA
  • Kinesin