Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2

We have previously described the biochemical isolation of 65 kDa and 120 kD a microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAP s have subsequently been shown to be structural MAPs that reconstitute 30 n m cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devise d a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the co ld stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa M APs are also associated with the cold-sensitive microtubules. On gradient g els, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC cla ss known to move to the plus ends of microtubules. Antibodies raised agains t specific expressed sequences recognize the upper band, while the lower ba nd is recognized by antibodies to the tobacco kinesin-related protein, TKRP 125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homolo gous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cort ical microtubules and the preprophase band, but anti-DcKRP120-2 does so onl y weakly. Both clearly stain the spindle and the phragmoplast, but in a pro portion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-li ne where the plus ends of the microtubules overlap. We discuss the potentia l roles of these proteins during the microtubule cycle.