Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.     

Genetically Controlled Self-Aggregation of Cell-Surface-Engineered Yeast Responding to Glucose Concentration

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and α-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agα1p (α-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and α-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.     

Pheromone-regulated Genes Required for Yeast Mating Differentiation

Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for β-galactosidase (β-gal) expression in the presence and absence of α factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: β-gal and Fig2::β-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.     

Cytoskeletal confinement of CX3CL1 limits its susceptibility to proteolytic cleavage by ADAM10

CX₃CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX₃CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX₃CL1. We observed that the majority of cell surface CX₃CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX₃CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX₃CL1 confinement and increased CX₃CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand.     

Evolution of the multidomain protein wheat germ agglutinin

Summary We compared the homologous amino acid sequences of hevein and each of the four domains (A, B, C, and D) of wheat germ agglutinin and used them to construct a pseudophylogenetic tree relating these sequences to a hypothetical common ancestor sequence. In the crystal structure of the wheat germ agglutinin dimer, six pseudo-twofold rotational symmetry axes have previously been located in addition to the true twofold axis. Four of these relate two nonidentical domains to each other in each of the four possible pairs constituting the sugar-binding sites (A₁D₂, A₂D₁, B₁C₂, and B₂C₁). The remaining two relate contiguous unique pairs of sugar-binding sites to each other (A₁D₂ to B₁C₂, and A₂D₁ to B₂C₁). These latter two sets of pairs are related to each other by the true twofold axis. Side chains that mediate sugar binding in the interfaces of each of the four pairs were found to be largely conserved. The sequence homology, taken together with these pseudo-symmetry elements in the dimer structure, suggests a pathway for the evolution of the four-domain molecule from a single-domain dimer that can be correlated with simultaneous development of the saccharide-binding sites.     

Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin

a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone. Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity. Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.     

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX₂CXC and twin CX₂C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.     

Secretion and surface display of green fluorescent protein using the yeast Saccharomyces cerevisiae

Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.     

Role of Fig2p in agglutination in Saccharomyces cerevisiae

In W303-derived strains, disruption of FIG2 increased agglutinability of α cells, but not a cells, and did not alter expression of α-agglutinin, binding of ¹²⁵I-labeled α-agglutinin, or mating efficiency. Fig2p overexpression led to α-cell-specific suppression of agglutinability. These results imply that Fig2p is an indirect masker of the active sites in α-agglutinin.     

Maintenance of mating cell integrity requires the adhesin Fig2p

Fungal adhesins represent a large family of serine/threonine-rich secreted glycoproteins. Adhesins have been shown to play roles in heterotypic and homotypic cell-cell adhesion processes, morphogenetic pathways and invasive/pseudohyphal growth, frequently in response to differentiation cues. Here we address the role of the Saccharomyces cerevisiae mating-specific adhesin Fig2p. Cells lacking FIG2 possess a variety of mating defects that relate to processes involving the cell wall, including morphogenetic defects, cell fusion defects, and alterations in agglutination activities. We found that mating-specific morphogenetic defects caused by the absence of FIG2 are suppressible by increased external osmolarity and that, during mating, fig2Δ cells display reduced viability relative to wild-type cells. These defects result from alterations in signaling activated by the mating and cell integrity pathways. Finally, we show that fig2Δ zygotes also have defects in zygotic spindle positioning that are osmoremedial, whereas the requirements for FIG2 in normal cell-cell agglutination and cell fusion during mating are insensitive to changes in the extracellular osmotic environment. We conclude that FIG2 performs distinct functions in the mating cell wall that are separable with respect to their ability to be suppressed by changes in external osmolarity and that a fundamental role of FIG2 in mating cells is the maintenance of cell integrity.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]₁₂ and [Hex]₈, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.     

Insights into the structure,function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX₃CX₂C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.     

FH3, A Domain Found in Formins, Targets the Fission Yeast Formin Fus1 to the Projection Tip During Conjugation

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1⁺ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.     

A Biochemical Guide to Yeast Adhesins: Glycoproteins for Social and Antisocial Occasions

Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.     

Genomic and Proteomic Analyses of the Agarolytic System Expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a β-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an α-neoagarobiose hydrolase to release galactose from agarose.     

Structure,Adsorption to Host,and Infection Mechanism of Virulent Lactococcal Phage p2

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k_off values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.     

A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions.

Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.