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© The Rockefeller University Press, 0021-9525/1998//1763 $5.00
The Journal of Cell Biology, Volume 143, Number 7, , 1998 1763-1774

Regular Articles

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1



Alexandra M. Ainsztein*, Stefanie E. Kandels-Lewis*, Alastair M. Mackay{ddagger}, and William C. Earnshaw*

* Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom; and {ddagger} Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21218

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hs{alpha}. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Key Words: centromeres • heterochromatin • HP1 • INCENP • mitosis

Abbreviations used in this paper: aa, amino acids; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione S-transferase; HP1Hs{alpha} and HP1Hs{gamma}, human heterochromatin protein {alpha} and {gamma}; INCENP, inner centromere protein; ONPG, o-nitrophenyl β-D-galactopyranoside.

These experiments were supported by a grant from the Wellcome Trust, of which W.C. Earnshaw is a Principal Research Fellow.

Alexandra M. Ainsztein's present address is Laboratory of Molecular Embryology, NICHD, Building 18, Room 106, 18 Library Dr., MSC-5431, Bethesda, MD 20892-5431. Alastair M. Mackay's present address is Osiris Therapeutics, Inc., 2001 Aliceana St., Baltimore, MD 21231-2001.


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