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© The Rockefeller University Press, 0021-9525/1998//1029 $5.00
The Journal of Cell Biology, Volume 143, Number 4, , 1998 1029-1040

Regular Articles

Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function



Christian Hofmann, Iain M. Cheeseman, Bruce L. Goode, Kent L. McDonald, Georjana Barnes, and David G. Drubin

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

Key Words: mitosis • microtubule • anaphase • cytoskeleton • spindle

Abbreviations used in this paper: DAPI, 4', 6-diamidino-2-phenylindole; 5-FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; GST, glutathione-S-transferase.

The Tub4p antibody was a generous gift from Tim Stearns. We thank Michelle (Shelly) Jones and Mark Winey for sharing results before publication.

Address all correspondence to David G. Drubin, 401 Barker Hall, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202. Tel.: (510) 642-3692. Fax: (510) 642-6420. E-mail: drubin{at}uclink4.berkeley.edu


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