INCENP centromere and spindle targeting: Identification of essential conserved motifs and involvement of heterochromatin protein HP1

The inner centromere protein (INCENP) has a modular organization, with doma ins required for chromosomal and cytoskeletal functions concentrated near t he amino and carboxyl termini, respectively. In this study we have identifi ed an autonomous centromere- and midbody-targeting module in the aminotermi nal 68 amino acids of INCENP. Within this module, we have identified two ev olutionarily conserved amino acid sequence motifs: a 13-amino acid motif th at is required for targeting to centromeres and transfer to the spindle, an d an Il-amino acid motif that is required for transfer to the spindle by mo lecules that have targeted previously to the centromere. To begin to unders tand the mechanisms of INCENP function in mitosis, we have performed a yeas t two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and het erochromatin protein HP1(Hs alpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromat in, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.