The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport.
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.
Mueller
Joshua
J
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Perrone
Catherine A
CA
Bower
Raqual
R
Cole
Douglas G
DG
Porter
Mary E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D015139:Blotting, Southern; D015153:Blotting, Western; D002503:Centromere / Q000648:ultrastructure; D002696:Chlamydomonas / Q000378:metabolism; D002923:Cilia / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D005407:Flagella / Q000502:physiology*; D008040:Genetic Linkage; D016547:Kinesins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D000939:Epitopes; D012313:RNA; D016547:Kinesins