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Published online
doi:10.1083/jcb.200605097
The Journal of Cell Biology, Vol. 176, No. 1, 51-63
The Rockefeller University Press, 0021-9525 $30.00
© Cai et al.
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Article

Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells



Dawen Cai1,2, Adam D. Hoppe3, Joel A. Swanson3, and Kristen J. Verhey2

1 Biophysics Research Division, 2 Department of Cell and Developmental Biology, and 3 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109

Correspondence to Kristen J. Verhey: kjverhey{at}umich.edu

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

Abbreviations used in this paper: DTNB, 3-carboxy-4-nitrophenyl disulfide 6,6'-dinitro-3,3'-dithiodibenzoic acid bis(3-carboxy-4-nitrophenyl) disulfide; FP, fluorescent protein; FRET, fluorescence resonance energy transfer; KHC, kinesin heavy chain; KLC, kinesin light chain; mCit, monomeric Citrine; TPR, tetratricopeptide repeat; SLO, streptolysin O.


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