Identification of a 40 x 10(3) Mr centromere-associated protein in cultured mammalian cells

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32-9, produced antibodies that reacted with a 40 x 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 x 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32-9 demonstrated that the 40 x 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 x 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 x 10(3) Mr protein remained associated with the insoluble cellular material. The 40 x 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 x 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.