Binding of a proline-independent hydrophobic motif by the Candida albicans Rvs167-3 SH3 domain.

Src-homology 3 (SH3) domains are small protein-protein interaction modules. While most SH3 domains bind to proline-x-x-proline (PxxP) containing motifs in their binding partners, some SH3 domains recognize motifs other than proline-based sequences. Recently, we showed that the SH3 domain of Candida albicans Rvs167-3 binds peptides enriched in hydrophobic residues and containing a single proline residue (RΦxΦxΦP, where x is any amino acid and Φ is a hydrophobic residue). Here, we demonstrate that the proline in this motif is not required for Rvs167-3 SH3 recognition. Through mutagenesis studies we show that binding of the peptide ligand involves the conserved tryptophan in the canonical PxxP binding pocket as well as residues in the extended n-Src loop of Rvs167-3 SH3. Our studies establish a novel, proline-independent, binding sequence for Rvs167-3 SH3 (RΦxΦxΦ) that is comprised of a positively charged residue (arginine) and three hydrophobic residues.

Evolution of the SH3 Domain Specificity Landscape in Yeasts.

To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.

Identification and characterization of Rvs162/Rvs167-3, a novel N-BAR heterodimer in the human fungal pathogen Candida albicans.

Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi.

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5(+)CD19(+) B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for ß-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. ß-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for ß-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to ß-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.

Role of the synthase domain of Ags1p in cell wall alpha-glucan biosynthesis in fission yeast.

The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.

Role of the alpha-glucanase Agn2p in ascus-wall endolysis following sporulation in fission yeast.

During sporulation in the ascomyceteous fungus Schizosaccharomyces pombe, diploid cells undergo differentiation into asci containing four haploid ascospores, which are highly resistant to environmental stresses. Although the morphogenetic processes involved in ascospore formation have been studied extensively, little is known about the molecular mechanism that ensures the release of mature ascospores from the ascus, allowing their dispersal into the environment. Recently, we identified Agn2p as the paralogue of the characterized endo-(1,3)-alpha-glucanase Agn1p, and observed that asci deleted for agn2 are defective in ascospore dispersal. Here, we focus on the cellular and biochemical functions of Agn2p. By placing agn2 under the control of an inducible promoter, we show that expression of agn2 is required for the efficient release of ascospores from their asci. Furthermore, we characterize the enzyme activity of purified recombinant Agn2p and show that Agn2p, like Agn1p, is an endo-(1,3)-alpha-glucanase that produces predominantly (1,3)-alpha-glucan pentasaccharides. Finally, we demonstrate that exogenous addition of purified Agn2p liberated the ascospores from asci deleted for agn2. We propose that Agn2p participates in the endolysis of the ascus wall by hydrolysing its (1,3)-alpha-glucan, thereby assisting in the release of ascospores.

Mechanism of action of the endo-(1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum.

(1-->3)-alpha-glucanases catalyze the hydrolysis of fungal cell wall (1-->3)-alpha-glucan, and function during cell division of yeasts containing this cell wall component or act in mycoparasitic processes. Here, we characterize the mechanism of action of the (1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum. We observed that MutAp releases predominantly beta-glucose upon hydrolysis of crystalline (1-->3)-alpha-glucan, indicating inversion of the anomeric configuration. After having identified (1-->3)-alpha-glucan tetrasaccharide as the minimal substrate for MutAp, we showed that reduced (1-->3)-alpha-glucan pentasaccharide is cleaved into a trisaccharide and a reduced disaccharide, demonstrating that MutAp displays endo-hydrolytic activity. We propose a model for the catalytic mechanism of MutAp, whereby the enzyme breaks an intrachain glycosidic linkage of (1-->3)-alpha-glucan, and then continues its hydrolysis towards the non-reducing end by releasing beta-glucose residues in a processive manner.

Transcription regulation of the alpha-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast.

During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1+, which encodes the alpha-glucanase Agn1p. The agn1 promoter contains three copies of the nucleotide sequence motif CCAGCC, whose presence seems to correlate with Ace2p-mediated transcription activation. Here, we describe a simple plate-based assay utilizing as a reporter the secreted glucoamylase of Arxula adeninivorans to investigate the function of this motif. We show that not all three repeats, but only the two most proximal to the transcription start point, act as an upstream activating sequence (UAS). Finally, we demonstrate that this UAS is essential for agn1 promoter activity in vivo.

The structure of cell wall alpha-glucan from fission yeast.

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.

Role of the alpha-glucanase Agn1p in fission-yeast cell separation.

Cell division in the fission yeast Schizosaccharomyces pombe yields two equal-sized daughter cells. Medial fission is achieved by deposition of a primary septum flanked by two secondary septa within the dividing cell. During the final step of cell division, cell separation, the primary septum is hydrolyzed by an endo-(1,3)-beta-glucanase, Eng1p. We reasoned that the cell wall material surrounding the septum, referred to here as the septum edging, also must be hydrolyzed before full separation of the daughter cells can occur. Because the septum edging contains (1,3)-alpha-glucan, we investigated the cellular functions of the putative (1,3)-alpha-glucanases Agn1p and Agn2p. Whereas agn2 deletion results in a defect in endolysis of the ascus wall, deletion of agn1 leads to clumped cells that remained attached to each other by septum-edging material. Purified Agn1p hydrolyzes (1,3)-alpha-glucan predominantly into pentasaccharides, indicating an endo-catalytic mode of hydrolysis. Furthermore, we show that the transcription factors Sep1p and Ace2p regulate both eng1 and agn1 expression in a cell cycle-dependent manner. We propose that Agn1p acts in concert with Eng1p to achieve efficient cell separation, thereby exposing the secondary septa as the new ends of the daughter cells.