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Vol. 19, Issue 7, 3124-3137, July 2008

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High-Resolution Crystal Structure and In Vivo Function of a Kinesin-2 Homologue in Giardia intestinalis

J. C. Hoeng^*,, S. C. Dawson^,, S. A. House, M. S. Sagolla, J. K. Pham, J. J. Mancuso, J. Löwe^*, and W. Z. Cande

*Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom; Section of Microbiology, University of California, Davis, Davis, CA 95616; and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3200

Submitted November 19, 2007; Revised April 16, 2008; Accepted April 29, 2008
Monitoring Editor: Tim Stearns

A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis–deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of G. intestinalis wild-type kinesin-2 (GiKIN2a) motor domain, with its docked neck linker and the hydrolysis-deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg²⁺ at 1.6 and 1.8 Å resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. G. intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes, with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in G. intestinalis.

These authors contributed equally to this work.

Address correspondence to: Scott Dawson scdawson{at}ucdavis.edu)

Abbreviations used: GiKIN2a, Giardia intestinalis kinesin 2a homolog; GiKIN2b, Giardia intestinalis kinesin 2b homolog; GiKIN2aT104N, ATP hydrolysis-deficient mutant strain of GiKIN2a gene; IFT, intraflagellar transport; DIC, differential interference contrast microscopy; KAP, kinesin-associated protein; GASP, novel Giardia coiled-coil protein; OSM-3, "osmotic avoidance-3" C. elegans kinesin-2 homolog that functions as a homodimer; pET17b, Novagen cloning vector; BL21 (DE3), E. coli strain for expression of recombinant proteins; IPTG, isopropyl β:D:1:thiogalactopyranoside; PEG, polyethylene glycol; CCP4, Collaborative Computational Project Number 4; IFT140, intraflagellar transport protein, Complex A, subunit 140; IFT81, intraflagellar transport protein, Complex B, subunit 81; GFP, green fluorescent protein; qPCR, quantitative PCR; His₆, histone 6–tagged protein; Ni-NTA, nickel-charged affinity resin for purification of His-tagged proteins.