THE PRODUCT OF THE SPINDLE FORMATION GENE SAD1(+) ASSOCIATES WITH THEFISSION YEAST SPINDLE POLE BODY AND IS ESSENTIAL FOR VIABILITY

Spindle formation in fission yeast occurs by the interdigitation of tw o microtubule arrays extending from duplicated spindle pole bodies whi ch span the nuclear membrane. By screening a bank of temperature-sensi tive mutants by anti-tubulin immunofluorescence microscopy, we previou sly identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lend.). 347:563-566). Here we describe the isolation and chara cterization of the sad1(+) gene. We show that the sad1.1 mutation affe cted both spindle formation and function. The sad1(+) gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical pheno types to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein mi grated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower t han its predicted mobility, and was exclusively associated with the sp indle pole body (SPB) throughout the mitotic and meiotic cycles. Micro tubule integrity was not required for Sad1 association with the SPB. U pon the differentiation of the SPB in metaphase of meiosis II, Sad1-st aining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end o f anaphase or kinetochores, we suggest that Sad1 is not a general comp onent of microtubule-interacting structures per se, but is an essentia l mitotic component that associates with the SPB but is not required f or microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.