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Vol. 16, Issue 3, 1341-1354, March 2005

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

Joshua Mueller ^* , Catherine A. Perrone ^* , Raqual Bower ^*, Douglas G. Cole , and Mary E. Porter ^*

^* Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455; Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052

Submitted October 27, 2004; Accepted December 10, 2004
Monitoring Editor: J. Richard McIntosh

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°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°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.

Abbreviations used: BAC, bacterial artificial chromosome; Dhc, dynein heavy chain; GFP, green fluorescent protein; HA, hemagglutin; IFT, intraflagellar transport; KAP, Kinesin-2 associated protein; LIC, light intermediate chain; M-N/5, minimal medium minus ammonium; PCD, primary ciliary dyskinesia; PKD, polycystic kidney disease; RFLP, restriction fragment length polymorphism; TAP, trisacetate phosphate.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

Address correspondence to: Mary E. Porter (porte001{at}umn.edu).

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