Carboxyl terminus of mitosin is sufficient to confer spindle pole localization

Mitosin is a nuclear protein of 3,113 amino acids which has been shown to associate with the mitotic apparatus, especially the kinetochore, during mitosis. In this paper we further confirmed its association with the spindle poles in normal monkey kidney CV1 cells by indirect immunofluorescence microscopy. When the carboxyl portion of mitosin containing amino acids 2,094-3,113 (named mitosin-pTN) was stably expressed in rat fibroblast Rat2 cells using a tetracycline-inducible system, strong spindle pole association was observed in addition to expected centromere localization. The same results were achieved in Chinese hamster ovary (CHO) cells. On the other hand, mitosin-pTC containing amino acids 2,756-3,113 was not targeted to spindle poles. Use of the FLAG epitope [Hopp et al., 1988] genetically fused to each amino terminus of these mutants eliminated possible artifacts due to antibody cross-reaction, since the spindle pole localization of wild-type mitosin was confirmed with a FLAG-tagged mutant by an antibody (anti-FLAG M2 monoclonal antibody) irrelevant to antibodies to mitosin. Our data also suggested a possible interaction of mitosin with the spindle microtubules. Interaction of mitosin with the major parts of the mitotic apparatus further implies an important role in mitosis.