Fractionation and characterization of kinesin II species in vertebrate brain.
Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.
Berezuk
Matthew A
MA
Department of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Schroer
Trina A
TA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000196:embryology* / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D002642:Chick Embryo; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000378:metabolism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D000072159:Dynactin Complex; D004398:Dyneins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000737:chemistry; D015151:Immunoblotting; D016547:Kinesins / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008870:Microtubules / Q000378:metabolism; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D010449:Peptide Mapping; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry
D002135:Calcium-Binding Proteins; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C074152:kinesin-II; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D004398:Dyneins; D016547:Kinesins