Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus.
A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.
Chan
J
J
Department of Cell Biology, John Innes Centre, Colney, Norwich, UK.
Rutten
T
T
Lloyd
C
C
D000818:Animals; D000906:Antibodies; D002467:Cell Nucleus / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D018552:Daucus carota / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013552:Swine; D014404:Tubulin / Q000378:metabolism
D000906:Antibodies; D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; D014404:Tubulin