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© The Rockefeller University Press, 0021-9525/1998//751 $5.00
The Journal of Cell Biology, Volume 143, Number 3, , 1998 751-765

Regular Articles

The Yeast Centrin, Cdc31p, and the Interacting Protein Kinase, Kic1p, Are Required for Cell Integrity



Donald S. Sullivan, Sue Biggins, and Mark D. Rose

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21– activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST–Kic1p and it bound to GST–Kic1p in gel overlay assays. Furthermore, GST–Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

Key Words: microtubule organizing center • spindle pole body • budding • actin • cell wall

Abbreviations used in this paper: 5-FOA, 5-fluoro-orotic acid; DAPI, 4',6'-diamidino-2-phenylindole; DIC, differential interference contrast; GST, glutathione-S-transferase; MAP kinase, mitogen-activated protein kinase; MTOC, microtubule organizing centers; PAK, p-21–activated kinases; SPB, spindle pole body; YPD, yeast extract with peptone and dextrose.

We are very grateful to J. Vossen and F. Klis for communication of results before publication. We thank J. Nickels, E. Lauzé, E. Weiss, and M. Winey for helpful discussions. We thank J. Goodhouse for assistance with the electron microscopy. We thank all members of the Rose lab for helpful discussions, especially C. Beh, A. Gammie, L. Satterwhite, and S. Clark. We thank I. Ivanovska, W. Khalfan, and A. Gammie for editorial comments on the manuscript. We are grateful to S. Fields, B. Deschenes, H. Sundberg, and T. Davis for strains and plasmids. We thank J. Kilmartin, T. Bretscher, and T. Wang for antibodies.

D. Sullivan's current address is Mitotix, Inc., One Kendall Square, Building 600, Cambridge, MA 02139.

S. Biggins's current address is Department of Physiology, University of California, San Francisco, CA 94143-0444.

Address all correspondence to Mark D. Rose, Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. Tel.: (609) 258-2804. Fax: (609) 258-6175. E-mail: mrose{at}molecular.princeton.edu


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