Single-molecule behavior of monomeric and heteromeric kinesins.
Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.
Pierce
D W
DW
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.
Hom-Booher
N
N
Otsuka
A J
AJ
Vale
R D
RD
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System; D019281:Dimerization; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009124:Muscle Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes
D002135:Calcium-Binding Proteins; D015801:Helminth Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C074152:kinesin-II; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins