A cdc-like autolytic Saccharomyces cerevisiae mutant altered in budding site selection is complemented by SPO12, a sporulation gene

Abstract

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lytJ-J allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37°C, lyti haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyti-Jllyti-l diploids failed to sporulate at both 24 and 37°C. Taking into account these characteristics, the lyti mutant could be considered a cdc-like mutant. By genetic transformation of an appropriate lyti strain with a genomic library, ligated to the multicopy vector YEpl3, we isolated a gene capable of complementing mitotic alterations but not the meiotic defect. This was the sporulation-specific gene SP012, which is expressed under the control of the locus ML4T in meiosis and is also expressed in the mitotic cycle (V. Parkes and L. H. Johnston, Nucleic Acids Res. 20:5617-5623, 1992). A significant level of SP012 mRNA can be detected when this gene is inserted in a multicopy plasmid.The isolation of fungal mutants displaying a thermosensitive autolytic phenotype is a general strategy to identify genes controlling cell wall integrity and cell viability. Mutants of this type have been described from Saccharomyces cerevisiae (6), Candida albicans (25), Schizosaccharomyces pombe (29), and Aspergillus nidulans (3). The characteristic shared by these mutant strains is loss of viability when grown at nonpermissive temperatures, leading to the release of the intracellular content. The wide variety of autolytic mutants that can be isolated reflects the complexity of functions involved in cell wall integrity and cell viability. However, some insight into the defect of the corresponding mutant can be obtained by analyzing complementation of the phenotypic defect by osmotic stabilization with sorbitol, which can be precisely determined by means of flow cytometry (10). Some mutants are osmotic remedial, suggesting that the phenotypic defect must be primarily located in the cell wall (25,36). Others are minimally complemented or not complemented at all by the osmotic stabilizer, an indication that the primary alteration probably does not concern cell wall functions, although eventually the cells release their intracellular content.One aspect of the autolytic mutants that has not been examined in detail is the relationship that they may have with the cell cycle. In some S. cerevisiae autolytic mutants, cell lysis occurs at a particular stage of the cell cy...

The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase.

“…The following evidence, along with the results obtained in this study, further implies the genetic interactions among TEMI, CDC15, LTE1, DBF2, and CDC5: (i) a high dose of SP012 suppressed teml-3, Itel, dbf2-1, and lytl-l (22,27,36); (ii) overexpression of CDC15 suppressed teml-3, dbf2-1, lytl-1, and cdc5-1 (15,22,36); (iii) multiple copies of CDC5 suppressed dbf2-1 and cdc15-1 (15); and (iv) multiple copies of TEM1 suppressed dbf2-2 (36). Assuming that Teml functions upstream of Cdc15, our results strongly support the presence of a cascade consisting of GTP-binding protein (Teml) and kinases (Cdcl5, Cdc5, and Dbf2) acting to terminate mitosis.…”

“…Moreover, these genes commonly interact with the SP012 gene. SP012, identified as a gene essential for sporulation (19), was isolated by its ability to suppress dbf2-1 (27) or lytl-l (22). We isolated the SP012 gene as a multicopy suppressor of teml-3 and cdcl5 (36) and found that the Itel disruption was also complemented by SP012 on a multicopy vector (36).…”

“…They are DBF2 (kinase), CDC5 (kinase), CDC14 (phosphatase), and LYT1 (unknown [22]). Among mutants in these genes, terminal phenotypes of the dbf2-2 cells are quite similar to those of the teml-defective cells and cdc15-2 cells (43); these mutant cells are arrested at telophase with high Hl-kinase activity under restrictive conditions.…”

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

“…There are a number of genes, encoding regulatory proteins, that, when mutated, cause a cell cycle arrest similar to mob1 mutants. There is an extensive array of genetic interactions linking them together, suggesting that they belong to a common pathway (Pringle and Hartwell, 1981;Wickner et al, 1987;Johnston et al, 1990;Parkes and Johnston, 1992;Kitada et al, 1993;Molero et al, 1993;Spevak et al, 1993;Keng et al, 1994;Shirayama et al, 1994aShirayama et al, , 1994bShirayama et al, , 1996Toyn and Johnston, 1994). We have begun to establish that MOB1 shares genetic interactions with several members of this class of genes.…”

A single‐copy suppressor of the Saccharomyces cerevisae late‐mitotic mutants cdc15 and dbf2 is encoded by the Candida albicansCDC14 gene

“…The S. cerevisiae strains used in the library screening and complementation assays were: L2C24d (cdc15-lyt1 ura3-52 MATa) (Molero et al, 1993); RH1779 (cdc14-1 ade2 his4 leu2 MATa) and L119-7d (dbf2 ura3-52 trp1+2 ade1 MATa), kindly supplied by Dr C. Kuhne; EO156 (tem1-3 ura3-52 leu2 his3 trp1) a gift from Dr A. Toh-e; 15DAU (cdc14-1 ura3 leu2 trp1 MATa), provided by Dr A. Bueno; 4955-3a (cdc16 his7 leu2 ura3 MATa) and 4086-23-2a (cdc23 his7 leu2 ura3 MATa) supplied by Dr M. Yuste; and WY46 (tem1::GAL-UPL-TEM1/Trp1 bar::hisG ade2-1 can1-1 his3-11, -15 leu2-3,-12 trp1-1 ura3-1 MATa) a gift from Dr R. J. Deshaies. The Candida albicans strains used were 1001 (Navarro-García et al, 1995) and CAI4 (Fonzi and Irwing, 1993).…”