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 wit h the spindle poles in normal monkey kidney CV1 cells by indirect immu nofluorescence microscopy. When the carboxyl portion of mitosin contai ning amino acids 2,094-3,113 (named mitosin-pTN) was stably expressed in rat fibroblast Rat2 cells using a tetracycline-inducible system, st rong spindle pole association was observed in addition to expected cen tromere localization. The same results were achieved in Chinese hamste r ovary (CHO) cells. On the other hand, mitosin-pTC containing amino a cids 2,756-3,113 was not targeted to spindle poles. Use of the FLAG ep itope [Hopp et al., 1988] genetically fused to each amino terminus of these mutants eliminated possible artifacts due to antibody cross-reac tion, since the spindle pole localization of wild-type mitosin was con firmed with a FLAG-tagged mutant by an antibody (anti-FLAG M2 monoclon al antibody) irrelevant to antibodies to mitosin. Our data also sugges ted a possible interaction of mitosin with the spindle microtubules. I nteraction of mitosin with the major parts of the mitotic apparatus fu rther implies an important role in mitosis. (C) 1997 Wiley-Liss, Inc.