C. Barroso et al., Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2, PLANT J, 24(6), 2000, pp. 859-868
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.