Protocol

Kinesin Protocols

Volume 164 of the series Methods in Molecular Biology™ pp 191-204

A Dominant Negative Approach for Functional Studies of the Kinesin II Complex

  • Vladimir I. GelfandAffiliated withDepartment of Cell and Structural Biology, University of Illinois at Urbana-Champaign
  • , Nathalie Le BotAffiliated withEuropean Molecular Biology Laboratory
  • , M. Carolina TumaAffiliated withDepartment of Molecular, Cellular, and Developmental Biology, Yale University
  • , Isabelle VernosAffiliated withEuropean Molecular Biology Laboratory

* Final gross prices may vary according to local VAT.

Get Access

Abstract

There are a few ways to generate a dominant negative mutant. Specifically for the study of functions of motor proteins, two approaches have been successfully used. The first approach consists of the deletion of the motor domain. In this case the mutant protein is not able to hydrolyze ATP or/and bind to the cytoskeleton, and consequently, unable to promote movement. This is often referred to as a “headless” mutant, as the motor domain is commonly referred as the “head” of a motor protein. This strategy has been used previously for the study of motor protein functions for both microtubule and actin-based motors (13). The second type of dominant negative mutant is a rigor mutant, generated by a point mutation in the ATP-binding site; as a result, the motor binds to the cytoskeletal filament (actin or microtubules) but cannot hydrolyze ATP; this ATP-bound form of the motor is irreversibly locked to its cytoskeleton partner, a state defined as a rigor complex. Rigor mutants have been first identified, sequenced, and characterized for the yeast kinesin-like motor Kar3p (4) and Drosophila melanogaster nod (5). In both cases, the mutant protein was shown to bind irreversibly to microtubules. These observations suggested the use of rigor mutants as a tool for investigating the function of kinesin-like motors, and this approach has been successfully used for conventional kinesin (6).

In order to generate a dominant negative mutant of kinesin II, we chose to use the “headless” approach for two reasons. First, it is known that kinesin II forms