Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h + cells and enhances cell adhesion when overexpressed.

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.

The involvement of Schizosaccharomyces pombe prm1 + in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1D mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1 + and the dni + genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cellcell contact region. In the prm1D mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1D nor prm1D dniD zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1D, and dni1D strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. M EMBRANE fusion is essential for several developmental processes. The characterization of mating in yeasts represents a useful tool for understanding the mechanisms involved in intercellular membrane merger and their regulation. In the fission yeast Schizosaccharomyces pombe, heterothallic cells belong to one of two mating types, M (h 2 cells) or P (h + cells) while h 90 strains are homothallic (Arcangioli and Thon 2004). In rich medium, h + and h 2 cells can proliferate actively together without mating. When nitrogen becomes scarce the cAMP level decreases, which triggers the expression of genes required for sexual differentiation. Cells arrest in G1 and polarize, giving rise to specialized cells termed shmoos (Yamamoto et al. 1997;Davey 1998;Nielsen 2004;Yamamoto 2004). After agglutination, the cross wall separating both parental cells is degraded, allowing cell fusion. Efficient cell fusion requires the correct organization of the cytoskeleton (Petersen et al. 1995(Petersen et al. , 1998aKurahashi et al. 2002;Doyle et al. 2009). The S. cerevisiae FUS1 and the S. pombe fus1 + genes have the same name and both mutants exhibit cell fusion defects during mating, leading to an accumulation of prezygotes (mating intermediates in which the mating partners have established a stable contact but have not yet fused). However, the corresponding proteins are not related; while Saccharomyces cerevisiae Fus1p is a membrane protein (Trueheart et al. 1987), S. pombe Fus1p ...

SummaryIn fungi, cell adhesion is required for flocculation, mating and virulence, and it is mediated by covalently bound cell wall proteins termed adhesins. Map4, an adhesin required for mating in Schizosaccharomyces pombe, is N-glycosylated and O-glycosylated, and is an endogenous substrate for the mannosyl transferase Oma4p. Map4 has a modular structure with an N-terminal signal peptide, a serine and threonine (S/T)-rich domain that includes nine repeats of 36 amino acids (rich in serine and threonine residues, but lacking glutamines), and a C-terminal DIPSY domain with no glycosylphosphatidyl inositol (GPI)-anchor signal. Map4 can be extracted from cell walls with SDS/mercaptoethanol sample buffer or with mild alkali solutions. After extensive extraction with hot sample buffer, no more protein can be released by b-glucanases or alkali. Additionally, none of the cysteine residues of the protein is required for its retention at the cell wall. These results show that Map4 is not directly bound to b-glucans and point to the existence of alkali-and SDS/mercaptoethanol-sensitive linkages between cell wall proteins. The N-terminal S/T-rich regions are required for cell wall attachment, but the C-terminal DIPSY domain is required for agglutination and mating in liquid and solid media.