A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos

Abstract

Pan-kinesin peptide antibodies (Cole, D. G., Cande, W. Z., Baskin, R. J., Skoufias, D. A., Hogan, C. J., and Scholey, J. M. (1992) J. Cell Sci. 101, 291-301; Sawin, K. E., Mitchinson, T. J., and Wordeman, L. G. (1992) J. Cell Sci. 101, 303-313) were used to identify and isolate kinesin-related proteins (KRPs) from Drosophila melanogaster embryonic cytosol. These KRPs cosedimented with microtubules (MTs) polymerized from cytosol treated with AMP-PNP (adenyl-5'-yl imidodiphosphate), and one of them, KRP130, was further purified from ATP eluates of the embryonic MTs. Purified KRP130 behaves as a homotetrameric complex composed of four 130-kDa polypeptide subunits which displays a "slow" plus-end directed motor activity capable of moving single MTs at 0.04 +/- 0.01 microns/s. The 130-kDa subunit of KRP130 was tested for reactivity with monoclonal and polyclonal antibodies that are specific for various members of the kinesin superfamily. Results indicate that the KRP130 subunit is related to Xenopus Eg5 (Sawin, K. E., Le Guellec, K. L., Philippe, M., Mitchinson, T. J. (1992) Nature 359, 540-543), a member of the BimC subfamily of kinesins. Therefore, KRP130 appears to be the first Drosophila KRP, and the first member of the BimC subfamily in any organism, to be purified from native tissue as a multimeric motor complex.

FIG. 1. Bio-Gel A-1.5m gel filtration of ATP-eluted Drorophila MT-binding proteins

A, Coomassie Blue-stained SDS gel. Lane L shows the ATP-eluted MAPs that were loaded onto the column. Duplicate immunoblots were probed with “LAGSE” antihody (similar results were obtained using other pankinesin antibodies 16, 34)) (B), anti-Eg5 antibody (C), and SUK4 antibody (D). The column (1.6 × 85 cm) was equilibrated with 100 μm ATP, 150 mm KCl in PMEG buffer. Fractions (3.0 ml) 23–42 are shown on the gels and blots here. The void volume corresponded to fraction 24, and the included volume corresponded to fraction 59.

FIG. 2. Fractionation of Drosophila kinesin and KRP₁₃₀ by sucrose density gradient centrifugation

A, KRP₁₃₀. Coomassie Blue-stained 5-20% acrylamide gradient SDS gel of a 5-20% sucrose gradient shows that KRP₁₃₀ sediments as a single 7.6 S peak. No detectable polypeptides greater than 20 kDa coelute with the 130-kDa polypeptide. B, kinesin (KHC and KLC). Coomassie Blue-stained 7.5% acrylamide SDS gel of 5-20% sucrose gradient shows that kinesin sediments as a single 9.1 S peak. Vertical arrowheads indicate that peak KRP₁₃₀ and kinesin fractions.

FIG. 3. Immunoreactivity of KRP₁₃₀ with anti-Eg5 antibody

A silver-stained SDS gel of a 5-20% sucrose gradient shows that KRP₁₃₀ sediments as a single peak. B, duplicate immunoblot probed with anti-Eg5 antibody. Vertical arrowheads indicate that peak KRP₁₃₀ fraction.