The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

Abstract

Abstract. 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. . Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad/+ gene. We show that the sadl.1 mutation affected both spindle formation and function. The sadl + 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 sadl.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sadl 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 Sadl association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sadl-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sadl led to association with the nuclear periphery. As Sadl was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sadl 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. Sadl 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.

Dissection of fission yeast microtubule associating protein p93Dis1: regions implicated in regulated localization and microtubule interaction

“…2c). This was verified in combination with immunofluorescence microscopy using anti-sad1 (Hagan & Yanagida 1995) and anti-tubulin antibodies (data not shown). The C polypeptide failed to bind to microtubule arrays, consistent with the in vitro result.…”

Large‐scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP‐fusion genomic DNA library

“…TB98 and TC80 also stained microtubule organizing centres other than the SPB, and additionally, they both stained the nuclear envelope. This may be a similar situation in which a wellcharacterized SPB component, sad1 protein, also localizes in the nuclear membrane when expressed on a multicopy plasmid (Hagan & Yanagida 1995). Five gene products were localized to microtubules (Table 2; Fig.…”

Role of the α‐glucanase Agn2p in ascus‐wall endolysis following sporulation in fission yeast

“…Following karyogamy, one round of DNA replication takes place, followed by two successive nuclear divisions, a reductional division (meiosis I) and an equational division (meiosis II), generating four haploid nuclei, which develop into individual ascospores (Tanaka and Hirata, 1982;Shimoda, 2004). Ascospore formation starts during meiosis II, when the morphology of the four nucleusassociated spindle pole bodies changes into a multi-layered structure (Hirata and Shimoda, 1994;Hagan and Yanagida, 1995). These specialized structures expand more or less synchronously by the fusion of membrane vesicles at each individual nucleus, generating double-membrane structures, termed the forespore membranes, which will encapsulate their respective nucleus (Tanaka and Hirata, 1982).…”