The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.
Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected 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 phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining 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 of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for 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.
Hagan
I
I
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Yanagida
M
M
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D002451:Cell Compartmentation; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D002467:Cell Nucleus / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D017353:Gene Deletion; D005800:Genes, Fungal / Q000235:genetics*; D005804:Genes, Lethal / Q000235:genetics; D008870:Microtubules / Q000502:physiology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D029702:Schizosaccharomyces pombe Proteins; D017422:Sequence Analysis, DNA; D008941:Spindle Apparatus / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C093512:SAD1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins