scholarly journals Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls

2020 ◽  
Vol 117 (36) ◽  
pp. 22061-22067
Author(s):  
Marian Samuel Vogt ◽  
Gesa Felicitas Schmitz ◽  
Daniel Varón Silva ◽  
Hans-Ulrich Mösch ◽  
Lars-Oliver Essen
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Molecular Model ◽  
Md Simulations ◽  
Binding Pocket ◽  
Fungal Cell ◽  
Final Destination ◽  
Fragment Screening

The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence

1987 ◽  
Vol 7 (9) ◽  
pp. 3306-3314
Author(s):  
S Silve ◽  
M Monod ◽  
A Hinnen ◽  
R Haguenauer-Tsapis
Keyword(s):  
Cell Wall ◽  
Acid Phosphatase ◽  
Gene Product ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
In Vitro Mutagenesis ◽  
Heterologous System ◽  
Pho5 Gene

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall glycoprotein that follows the yeast secretory pathway. We used in vitro mutagenesis to construct a deletion (delta SP) including the entire signal sequence and four amino acids of the mature sequence of APase. An APase-deficient yeast strain was transformed with a high-copy-number plasmid carrying the PHO5/delta SP gene. When expressed in vivo, the PHO5/delta SP gene product accumulated predominantly as an inactive, unglycosylated form located inside the cell. A large part of this unglycosylated precursor underwent proteolytic degradation, but up to 30% of it was translocated, core glycosylated, and matured by the addition of mannose residues, before reaching the cell wall. It appears, therefore, that the signal sequence is important for efficient translocation and core glycosylation of yeast APase but that it is not absolutely necessary for entry of the protein into the yeast secretory pathway. mRNA obtained by in vitro transcription of PHO5 and PHO5/delta SP genes were translated in vitro in the presence of either reticulocyte lysate and dog pancreatic microsomes or yeast lysate and yeast microsomes. The PHO5 gene product was translocated and core glycosylated in the heterologous system and less efficiently in the homologous system. We were not able to detect any translocation or glycosylation of PHO5/delta SP gene product in the heterologous system, but a very small amount of core suppression of glycosylated material could be evidenced in the homologous system.

scholarly journals Laccase Expression in Murine Pulmonary Cryptococcus neoformans Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 3124-3127 ◽  
Author(s):  
Javier Garcia-Rivera ◽  
Stephanie C. Tucker ◽  
Marta Feldmesser ◽  
Peter R. Williamson ◽  
Arturo Casadevall
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Lung Tissue ◽  
Immunogold Electron Microscopy ◽  
Cell Cytoplasm ◽  
Fungal Cell ◽  
Time Of Infection

ABSTRACT Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

scholarly journals In vitro and in vivo reactivity to fungal cell wall agents in sarcoidosis

2011 ◽  
Vol 166 (1) ◽  
pp. 87-93 ◽  
Author(s):  
M. Terčelj ◽  
S. Stopinšek ◽  
A. Ihan ◽  
B. Salobir ◽  
S. Simčič ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fungal Cell Wall ◽  
Fungal Cell

scholarly journals Up against the Wall: Is Yeast Cell Wall Integrity Ensured by Mechanosensing in Plasma Membrane Microdomains?

2014 ◽  
Vol 81 (3) ◽  
pp. 806-811 ◽  
Author(s):  
Christian Kock ◽  
Yves F. Dufrêne ◽  
Jürgen J. Heinisch
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Yeast Cell ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Yeast Cell Wall ◽  
Membrane Microdomains ◽  
Wall Structure ◽  
Fungal Cell ◽  
Membrane Spanning

ABSTRACTYeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

Sterol trafficking between the endoplasmic reticulum and plasma membrane in yeast

2006 ◽  
Vol 34 (3) ◽  
pp. 356-358 ◽  
Author(s):  
D.P. Sullivan ◽  
H. Ohvo-Rekilä ◽  
N.A. Baumann ◽  
C.T. Beh ◽  
A.K. Menon
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Wild Type Strain ◽  
Binding Pocket ◽  
Oxysterol Binding Protein ◽  
Sterol Transport ◽  
Transport Cycle ◽  
Independent Mechanism

We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816–5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154–158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.

scholarly journals Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

2014 ◽  
Vol 13 (12) ◽  
pp. 1484-1493 ◽  
Author(s):  
Julie M. Wolf ◽  
Javier Espadas-Moreno ◽  
Jose L. Luque-Garcia ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cryptococcus Neoformans ◽  
Extracellular Vesicles ◽  
Protein Composition ◽  
Multivesicular Body ◽  
Dense Matrix ◽  
Fungal Cell ◽  
Content Type ◽  
Pass Through

ABSTRACTCryptococcus neoformansproduces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.

scholarly journals Fluconazole and Lipopeptide Surfactin Interplay During Candida albicans Plasma Membrane and Cell Wall Remodeling Increases Fungal Immune System Exposure

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 314 ◽  
Author(s):  
Jakub Suchodolski ◽  
Daria Derkacz ◽  
Jakub Muraszko ◽  
Jarosław J. Panek ◽  
Aneta Jezierska ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Immune System ◽  
Antifungal Drugs ◽  
Stable Complex ◽  
Host Immune System ◽  
Chitin Synthesis ◽  
Fungal Cell ◽  
In Silico Studies

Recognizing the β-glucan component of the Candida albicans cell wall is a necessary step involved in host immune system recognition. Compounds that result in exposed β-glucan recognizable to the immune system could be valuable antifungal drugs. Antifungal development is especially important because fungi are becoming increasingly drug resistant. This study demonstrates that lipopeptide, surfactin, unmasks β-glucan when the C. albicans cells lack ergosterol. This observation also holds when ergosterol is depleted by fluconazole. Surfactin does not enhance the effects of local chitin accumulation in the presence of fluconazole. Expression of the CHS3 gene, encoding a gene product resulting in 80% of cellular chitin, is downregulated. C. albicans exposure to fluconazole changes the composition and structure of the fungal plasma membrane. At the same time, the fungal cell wall is altered and remodeled in a way that makes the fungi susceptible to surfactin. In silico studies show that surfactin can form a complex with β-glucan. Surfactin forms a less stable complex with chitin, which in combination with lowering chitin synthesis, could be a second anti-fungal mechanism of action of this lipopeptide.

Overview of the Interplay Between Cell Wall Integrity Signaling Pathways and Membrane Lipid Biosynthesis in Fungi: Perspectives forAspergillus fumigatus

2020 ◽  
Vol 21 (3) ◽  
pp. 265-283 ◽  
Author(s):  
João Henrique T.M. Fabri ◽  
Marina C. Rocha ◽  
Iran Malavazi
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Signaling Pathways ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Pathogenic Fungi ◽  
Cell Wall Integrity ◽  
Immune Recognition ◽  
Fungal Cell ◽  
Low Efficiency

:The cell wall (CW) and plasma membrane are fundamental structures that define cell shape and support different cellular functions. In pathogenic fungi, such as Aspegillus fumigatus, they not only play structural roles but are also important for virulence and immune recognition. Both the CW and the plasma membrane remain as attractive drug targets to treat fungal infections, such as the Invasive Pulmonary Aspergillosis (IPA), a disease associated with high morbimortality in immunocompromised individuals. The low efficiency of echinocandins that target the fungal CW biosynthesis, the occurrence of environmental isolates resistant to azoles such as voriconazole and the known drawbacks associated with amphotericin toxicity foster the urgent need for fungal-specific drugable targets and/or more efficient combinatorial therapeutic strategies. Reverse genetic approaches in fungi unveil that perturbations of the CW also render cells with increased susceptibility to membrane disrupting agents and vice-versa. However, how the fungal cells simultaneously cope with perturbation in CW polysaccharides and cell membrane proteins to allow morphogenesis is scarcely known. Here, we focus on current information on how the main signaling pathways that maintain fungal cell wall integrity, such as the Cell Wall Integrity and the High Osmolarity Glycerol pathways, in different species often cross-talk to regulate the synthesis of molecules that comprise the plasma membrane, especially sphingolipids, ergosterol and phospholipids to promote functioning of both structures concomitantly and thus, cell viability. We propose that the conclusions drawn from other organisms are the foundations to point out experimental lines that can be endeavored in A. fumigatus.

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