THE SPINDLE POLE BODY COMPONENT SPC97P INTERACTS WITH THE GAMMA-TUBULIN OF SACCHAROMYCES-CEREVISIAE AND FUNCTIONS IN MICROTUBULE ORGANIZATION AND SPINDLE POLE BODY DUPLICATION

Previously, me have shown that the gamma-tubulin Tub4p and the spindle pole body component Spc98p are involved in microtubule organization b y the yeast microtubule organizing centre, the spindle pole body (SPB) . In this paper we report the identification of SPC97 encoding an esse ntial SPB component that is in association with the SPB substructures that organize the cytoplasmic and nuclear microtubules. Evidence is pr ovided for a physical and functional interaction between Tub4p, Spc98p and Spc97p: first, temperature-sensitive spc97(ts) mutants are suppre ssed by high gene dosage of SPC98 or TUB4. Second, Spc97p interacts wi th Spc98p and Tub4p in the two-hybrid system. Finally, immunoprecipita tion and fractionation studies revealed complexes containing Tub4p, Sp c98p and Spc97p. Further support for a direct interaction of Tub4p, Sp c98p and Spc97p comes from the toxicity of strong SPC97 overexpression which is suppressed by co-overexpression of TUB4 or SPC98. Analysis o f temperature-sensitive spc97(ts) alleles revealed multiple spindle de fects. While spc97-14 cells are either impaired in SPB separation or m itotic spindle formation, spc97-20 cells show an additional defect in SPB duplication. We discuss a model in which the Tub4p-Spc98p-Spc97p c omplex is part of the microtubule attachment site at the SPB.