During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lyric enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a sporeautonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.

Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.z 1999 Federation of European Biochemical Societies.

The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtr1p (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane. DTR1 was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtr1p, a member of the poorly characterized DHA12 (drug:H ؉ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtr1p may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtr1p renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtr1p is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.Diploid a/␣ cells of the budding yeast Saccharomyces cerevisiae undergo a specialized developmental program termed sporulation when transferred to a nitrogen-free medium containing potassium acetate as nonfermentable carbon source. The final product of sporulation is an ascus that consists of four haploid spores surrounded by the ascus wall, the former vegetative cell wall (for a review, see reference 27). Spores are protected from adverse environmental conditions by the spore wall. Especially the surface layers contribute both to the spores' mechanical rigidity and their resistance against chemical and enzymatic attack (3). Spore wall synthesis begins with the formation of the prospore membrane, a bilayered electrondense structure that starts to form during the second meiotic division on the cytoplasmic side of each of the four spindle pole bodies by fusion of secretory vesicles (9, 13, 23, 31-33). As meiosis progresses, the prospore membrane extends along the outer surface of the nuclear envelope. Septins, among them the sporulation-specific proteins Spr3p and Spr28p, localize at the leading tip of the prospore membrane under control of the Gip1p-Glc7p phosphatase complex and might be involved in its extension and directed growth (14,16,44). Other proteins localized to the growing prospore membrane are SspIp, Ady3p, and Don1p, which form the leading edge protein coat (23,33) and presumably Sps2p (11,38), but th...

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J. Industr. Microbiol. 14, 523–530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle‐shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542–549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution. © 1997 John Wiley & Sons, Ltd.

Five cases of lipid-rich carcinomas of the breast were investigated ultrastructurally and immunohistochemically for alpha-lactalbumin (ALA), lactoferrin (Lfr) and human milk fat globule membrane antigen (HMFG-2). Staining for ALA and Lfr showed intensive reaction on nearly all of the tumour cells whereas immunoreaction for HMFG-2 revealed positivity in single cells. All tumours were negative for steroid receptor content. Ultrastructurally the tumour cells showed numerous intracytoplasmic non-membrane bound lipid droplets which were often found within autophagocytic vacuoles. Neither rough endoplasmic reticulum nor Golgi complexes showed any sign of lipid synthesis. Extrusion of lipid droplets and extracellular lipid deposition was not observed. In conclusion, our findings do not justify the consideration of lipid-rich carcinoma of the breast as a clearly defined group of tumours with specific secretory activity. Therefore, the term lipid-rich carcinoma should be used in preference to lipid-secreting, unless there is evidence of active lipid secretion.