Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3.
Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.
Adio
Sarah
S
Institute for Cell Biology, Ludwig-Maximilians-University Munich, Schillerstrasse 42, D-80336 Munich, Germany and Department of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
Bloemink
Marieke
M
Hartel
Michaela
M
Leier
Sven
S
Geeves
Michael A
MA
Woehlke
Günther
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D016547:Kinesins