Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.Comment: This work was supported by National Institutes of Health (NIH) Predoctoral Traineeship GM07232 and a University of California at Berkeley MacArthur and Lakhan-Pal Graduate Fellowship to K.L.L., Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund to AE-A, Marie Sklodowska-Curie Action H2020-MSCA-IF-2016 InsiliCardio, GA 75083 to CMA, and NIH R01 research grant GM21841 to J

Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.Comment: This work was supported by Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund and by National Institutes of Health (NIH) R01 Research Grant GM21841. Additionally, this project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Action H2020-MSCA-IF-2016 InsiliCardio, GA No. 75083

Multi-layered ZIF-coated cells for the release of bioactive molecules in hostile environments

Published on 01 August 2022Metal-organic framework (MOF) coatings on cells enhance viability in cytotoxic environments. Here, we show how protective multi-layered MOF bio-composite shells on a model cell system (yeast) enhance the proliferation of living cells exposed to hostile protease-rich environments via the dissolution of the shells and release of a protease inhibitor (antitrypsin).Lei Gan, Miriam de J. Velásquez-Hernández, Anita Emmerstorfer-Augustin, Peter Wied, Heimo Wolinski, Simone Dal Zilio, Marcello Solomon, Weibin Liang, Christian Doonan, and Paolo Falcar

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Energieeffizienz moderner Heizungsanlagen und deren Wirtschaftlichkeit.

Ziel dieser Arbeit ist es, die Vorteilhaftigkeit einer modernen Heizungsanlage zu veranschaulichen. Beginnend bei der Erklärung der verschiedenen Heizungsanlagen soll gezeigt werden, dass sich auch hohe Anschaffungskosten auf Grund der hier hohen Energieeffizienz rasch amortisieren. Basierend auf den hier errechneten Werte und anhand der vorherrschenden Gegebenheiten kann somit eine optimale Wahl der Heizungsanlage getroffen werden

Ladeinfrastruktur und die Problematik in der Elektromobilität

Bei dieser Arbeit soll die Problematik der Ladeinfrastruktur von Elektromobilität veranschaulicht werden. Am Anfang werden die verschiedensten Ladetechniken gezeigt. Weiteres wird auch die Ladeinfrastruktur für den privaten und öffentlichen Bereich betrachtet. Hierbei soll der Aufbau einer Ladestation erklärt werden, die dadurch resultierenden Erkenntnisse dienen der Veranschaulichung wie eine solche Ladestation am sinnvollsten umgesetzt werden kann

Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging.

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7

Bio-Polyester/Rubber Compounds: Fabrication, Characterization, and Biodegradation

Biobased and biodegradable polymers (BBDs) such as poly(3-hydroxy-butyrate), PHB, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are considered attractive alternatives to fossil-based plastic materials since they are more environmentally friendly. One major problem with these compounds is their high crystallinity and brittleness. In order to generate softer materials without using fossil-based plasticizers, the suitability of natural rubber (NR) as an impact modifier was investigated in PHBV blends. Mixtures with varying proportions of NR and PHBV were generated, and samples were prepared by mechanical mixing (roll mixer and/or internal mixer) and cured by radical C–C crosslinking. The obtained specimens were investigated with respect to their chemical and physical characteristics, applying a variety of different methods such as size exclusion chromatography, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermal analysis, XRD, and mechanical testing. Our results clearly indicate that NR–PHBV blends exhibit excellent material characteristics including high elasticity and durability. Additionally, biodegradability was tested by applying heterologously produced and purified depolymerases. pH shift assays and morphology analyses of the surface of depolymerase-treated NR–PHBV through electron scanning microscopy confirmed the enzymatic degradation of PHBV. Altogether, we prove that NR is highly suitable to substitute fossil-based plasticizers; NR–PHBV blends are biodegradable and, hence, should be considered as interesting materials for a great number of applications

Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2.

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo