Properties of the Kinesin‐3 NcKin3 motor domain and implications for neck function

Abstract

Members of the Kinesin‐3 family are microtubule motors involved in the transport of membranous cargo. NcKin3 from the fungus Neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. To determine how one of the heads is inactivated, we investigated truncated monomeric constructs. None of the constructs generated processive single‐molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. The kinetic analysis suggests futile ATP hydrolysis cycles, because a representative monomer showed a faster ATP turnover than the dimer while supporting slower motility. The K0.5,MT was 70‐fold lower, the microtubule‐bound portion of the kinetic cycle eight‐fold longer and the microtubule detachment rate almost 15‐fold slower than that of the dimer. Moreover, the monomer’s microtubule‐dependent ADP release occurred three‐fold to four‐fold faster than kcat (125 versus 34 s−1), whereas phosphate release was approximately equally fast (29 s−1). A dimeric construct containing a structure‐breaking insert between motor head and neck showed a similar behaviour. These data suggest that the heads of the wild‐type NcKin3 motor are strictly coupled via the neck domain, and that the dimeric structure is required for proper detachment after one ATPase cycle. This is the first direct comparison of a monomeric Kinesin‐3 with its dimeric full‐length counterpart, and the kinetic changes observed here may also apply to other Kinesin‐3 motors.

The Effect of Temperature on Microtubule-Based Transport by Cytoplasmic Dynein and Kinesin-1 Motors

“…Previous variable-temperature in vitro motility assays with kinesins (8)(9)(10) have been limited to a relatively narrow temperature window that roughly covers the survival range of most mammals. Broader ranges have been explored via biochemical methods (11,12), but not biophysical ones. Although published data on kinesin is somewhat limited, scant variable temperature data are available for cytoplasmic dynein.…”

The Aspergillus nidulans Kinesin-3 Tail Is Necessary and Sufficient to Recognize Modified Microtubules

“…In contrast, kinesin 3 from Neurospora crassa, NcKin3, was shown to be dimeric, but inactivates one of its motor heads to generate non-processive motility [17]. The data of Adio and Woehlke confirmed that the neck domain is required for dimerization and is essential for NcKin3 function: the absence of the neck altered the kinetic cycle fundamentally [18].…”

Interactions between Subunits in Heterodimeric Ncd Molecules

“…NcKin3 is a dimeric plusend-directed motor. The amino acid sequences of the heads are identical, but only one of the motor domains participates in hydrolyzing ATP and MT binding (7,44). The x-ray structure of the NcKin3 motor domain did not reveal any unusual features that could be linked to this unusual behavior of the motor domains (8).…”