Characterization of the Biophysical and Motility Properties of Kinesin from the Fungus Neurospora crassa

Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.