Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells.
As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.
Asada
T
T
Department of Biology, Faculty of Science, Osaka University, Japan.
Shibaoka
H
H
D000251:Adenosine Triphosphatases / Q000032:analysis; D002478:Cells, Cultured; D020558:GTP Phosphohydrolases / Q000032:analysis; D018715:Microscopy, Video; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000502:physiology*; D008870:Microtubules / Q000502:physiology*; D009068:Movement / Q000502:physiology*; D015388:Organelles / Q000737:chemistry / Q000502:physiology*; D010507:Periodicity; D010947:Plants, Toxic; D011485:Protein Binding; D013347:Subcellular Fractions; D013692:Telophase / Q000502:physiology*; D014026:Nicotiana / Q000166:cytology / Q000502:physiology*
D008869:Microtubule-Associated Proteins; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases