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  • 1.
    Alvarez, Francisco J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ryman, Kicki
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hooijmaijers, Cornelis
    Bulone, Vincent
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 8, p. 2770-2780Article in journal (Refereed)
    Abstract [en]

    The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

  • 2. Boban, Mirta
    et al.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Foisner, Roland
    Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 4, p. 2489-2495Article in journal (Refereed)
    Abstract [en]

    Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.

  • 3. Boban, Mirta
    et al.
    Pantazopoulou, Marina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Schick, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Foisner, Roland
    A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation2014In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 16, p. 3603-3613Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

  • 4. Chernomoretz, Ariel
    et al.
    Stolovitzky, Gustavo
    Labaj, Pawel P.
    Graf, Alexandra B.
    Darling, Aaron
    Burke, Catherine
    Noushmehr, Houtan
    Moraes, Milton Ozorio
    Dias-Neto, Emmanuel
    Guo, Yongli
    Xie, Zhi
    Lee, Patrick
    Shi, Leming
    Ruiz-Perez, Carlos A.
    Mercedes Zambrano, Maria
    Siam, Rania
    Ouf, Amged
    Richard, Hugues
    Lafontaine, Ingrid
    Wieler, Lothar H.
    Semmler, Torsten
    Ahmed, Niyaz
    Prithi-viraj, Bharath
    Nedunuri, Narasimha
    Mehr, Shaadi
    Banihashemi, Kambiz
    Lista, Florigio
    Anselmo, Anna
    Suzuki, Haruo
    Kuroda, Makoto
    Yamashita, Riu
    Sato, Yukoto
    Kaminuma, Eli
    Alpuche Aranda, Celia M.
    Martinez, Jesus
    Dada, Christopher
    Dybwad, Marius
    Oliveira, Manuela
    Schuster, Stephan
    Siwo, Geoffrey H.
    Jang, Soojin
    Seo, Sung Chul
    Hwang, Sung Ho
    Ossowski, Stephan
    Bezdan, Daniela
    Chaker, Salama
    Chatziefthimiou, Aspassia D.
    Udekwu, Klas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sezerman, Ugur
    Meydan, Cem
    Elhaik, Eran
    Gonnet, Gaston
    Schriml, Lynn M.
    Mongodin, Emmanuel
    Huttenhower, Curtis
    Gilbert, Jack
    Mason, Christopher E.
    Eisen, Jonathan
    Hirschberg, David
    Hernandez, Mark
    The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium inaugural meeting report2016In: Microbiome, ISSN 0026-2633, E-ISSN 2049-2618, Vol. 4, article id UNSP 24Article in journal (Refereed)
    Abstract [en]

    The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium is a novel, interdisciplinary initiative comprised of experts across many fields, including genomics, data analysis, engineering, public health, and architecture. The ultimate goal of the MetaSUB Consortium is to improve city utilization and planning through the detection, measurement, and design of metagenomics within urban environments. Although continual measures occur for temperature, air pressure, weather, and human activity, including longitudinal, cross-kingdom ecosystem dynamics can alter and improve the design of cities. The MetaSUB Consortium is aiding these efforts by developing and testing metagenomic methods and standards, including optimized methods for sample collection, DNA/RNA isolation, taxa characterization, and data visualization. The data produced by the consortium can aid city planners, public health officials, and architectural designers. In addition, the study will continue to lead to the discovery of new species, global maps of antimicrobial resistance (AMR) markers, and novel biosynthetic gene clusters (BGCs). Finally, we note that engineered metagenomic ecosystems can help enable more responsive, safer, and quantified cities.

  • 5.
    Davis, Monica M.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Alvarez, Francisco J.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Ryman, Kicki
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Holm, Åsa A.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wild-Type Drosophila melanogaster as a Model Host to Analyze Nitrogen Source Dependent Virulence of Candida albicans2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, p. e27434-Article in journal (Refereed)
    Abstract [en]

    The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.

  • 6. Khmelinskii, Anton
    et al.
    Blaszczak, Ewa
    Pantazopoulou, Marina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fischer, Bernd
    Omnus, Deike J.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Le Dez, Gaelle
    Brossard, Audrey
    Gunnarsson, Alexander
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barry, Joseph D.
    Meurer, Matthias
    Kirrmaier, Daniel
    Boone, Charles
    Huber, Wolfgang
    Rabut, Gwenael
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Knop, Michael
    Protein quality control at the inner nuclear membrane2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 516, no 7531, p. 410-+Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression(1). The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asicomplex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer(5), we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

  • 7.
    Ljungdahl, Per O
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Amino-acid-induced signalling via the SPS-sensing pathway in yeast.2009In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, no Pt 1, p. 242-7Article in journal (Refereed)
    Abstract [en]

    Yeast cells rely on the SPS-sensing pathway to respond to extracellular amino acids. This nutrient-induced signal transduction pathway regulates gene expression by controlling the activity of two redundant transcription factors: Stp1 and Stp2. These factors are synthesized as latent cytoplasmic proteins with N-terminal regulatory domains. Upon induction by extracellular amino acids, the plasma membrane SPS-sensor catalyses an endoproteolytic processing event that cleaves away the regulatory N-terminal domains. The shorter forms of Stp1 and Stp2 efficiently target to the nucleus, where they bind and activate transcription of selected genes encoding a subset of amino acid permeases that function at the plasma membrane to catalyse the transport of amino acids into cells. In the present article, the current understanding of events in the SPS-sensing pathway that enable external amino acids to induce their own uptake are reviewed with a focus on two key issues: (i) the maintenance of Stp1 and Stp2 latency in the absence of amino acid induction; and (ii) the amino-acid-induced SPS-sensor-mediated proteolytic cleavage of Stp1 and Stp2.

  • 8.
    Ljungdahl, Per O.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Daignan-Fornier, Bertrand
    Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae2012In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 190, no 3, p. 885-929Article in journal (Refereed)
    Abstract [en]

    Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra-and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear.

  • 9. Llopis-Torregrosa, Vicent
    et al.
    Vaz, Catarina
    Monteoliva, Lucia
    Ryman, Kicki
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Gacser, Attila
    Gil, Concha
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sychrova, Hana
    Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 7529Article in journal (Refereed)
    Abstract [en]

    The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1 Delta cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1 Delta mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

  • 10.
    Martins, António
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Pfirrmann, Thorsten
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Heessen, Stijn
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sundqvist, Gustav
    Bulone, Vincent
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 22, p. 8362-8378Article in journal (Refereed)
    Abstract [en]

    Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.

  • 11.
    Martins, António
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ring, Andreas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Omnus, Deike J.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Heessen, Stijn
    Pfirrmann, Thorsten
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling proteaseIn: Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5)-sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy-terminal of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Δtether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non- and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.

  • 12.
    Martins, António
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ring, Andreas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Omnus, Deike J.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Heessen, Stijn
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Pfirrmann, Thorsten
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Martin-Luther University Halle-Wittenberg, Germany..
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor-controlled Ssy5 signaling protease2019In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 30, no 21, p. 2709-2720Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5) sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy terminus of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Delta tether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution, and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non-and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.

  • 13.
    Omnus, Deike J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Latency of Transcription Factor Stp1 Depends on a Modular Regulatory Motif that Functions as Cytoplamsic Retention Determinant and Nuclear DegronManuscript (preprint) (Other academic)
  • 14.
    Omnus, Deike J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron2014In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, no 23, p. 3823-3833Article in journal (Refereed)
    Abstract [en]

    The Ssy1-Ptr3-Ssy5 (SPS)-sensing pathway enables yeast to respond to extracellular amino acids. Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation. The negative regulatory mechanisms impinging on the N-terminal domain are poorly understood. However, Stp1 latency depends on three inner nuclear membrane proteins, Asi1, Asi2, and Asi3. We report that the N-terminal domain of Stp1 contains a small motif, designated RI, that fully accounts for latency. RI is modular, mediates interactions with the plasma membrane, and can retain histone Htb2 in the cytoplasm. A novel class of STP1 mutations affecting RI were isolated that are less efficiently retained in the cytoplasm but remain under tight negative control by the Asi proteins. Intriguingly, these mutant proteins exhibit enhanced stability in strains lacking ASI1. Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.

  • 15.
    Omnus, Deike J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I2013In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 24, no 9, p. 1480-1492Article in journal (Refereed)
    Abstract [en]

    Ligand-induced conformational changes of plasma membrane receptors initiate signals that enable cells to respond to discrete extracellular cues. In response to extracellular amino acids, the yeast Ssy1-Ptr3-Ssy5 sensor triggers the endoproteolytic processing of transcription factors Stp1 and Stp2 to induce amino acid uptake. Activation of the processing protease Ssy5 depends on the signal-induced phosphorylation of its prodomain by casein kinase I (Yck1/2). Phosphorylation is required for subsequent Skp1/Cullin/Grr1 E3 ubiquitin ligase-dependent polyubiquitylation and proteasomal degradation of the inhibitory prodomain. Here we show that Rts1, a regulatory subunit of the general protein phosphatase 2A, and Ptr3 have opposing roles in controlling Ssy5 prodomain phosphorylation. Rts1 constitutively directs protein phosphatase 2A activity toward the prodomain, effectively setting a signaling threshold required to mute Ssy5 activation in the absence of amino acid induction. Ptr3 functions as an adaptor that transduces conformational signals initiated by the Ssy1 receptor to dynamically induce prodomain phosphorylation by mediating the proximity of the Ssy5 prodomain and Yck1/2. Our results demonstrate how pathway-specific and general signaling components function synergistically to convert an extracellular stimulus into a highly specific, tuned, and switch-like transcriptional response that is critical for cells to adapt to changes in nutrient availability.

  • 16.
    Omnus, Deike J.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Pfirrmann, Thorsten
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Andréasson, Claes
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy52011In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 15, p. 2754-2765Article in journal (Refereed)
    Abstract [en]

    Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.

  • 17.
    Pantazopoulou, Marina
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Boban, M.
    Foisner, R.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cdc48 and Ubx1/Shp1 are components of a novel inner nuclear membrane associated degradative pathway that governs the stability of Asi1Manuscript (preprint) (Other academic)
  • 18.
    Pantazopoulou, Marina
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Boban, Mirta
    Foisner, Roland
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation2016In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 129, no 20, p. 3770-3780Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t(1/2)<= 30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.

  • 19.
    Pfirrmann, Thorsten
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Heessen, Stijn
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Omnus, Deike J.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Andréasson, Claes
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp12010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 13, p. 3299-309Article in journal (Refereed)
    Abstract [en]

    Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells

  • 20. Pfirrmann, Thorsten
    et al.
    Lokapally, Ashwin
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hollemann, Thomas
    SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, article id e64870Article in journal (Refereed)
    Abstract [en]

    Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

  • 21.
    Ring, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Martins, António
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions2019In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 20, no 10, p. 775-784Article in journal (Refereed)
    Abstract [en]

    Evidence from multiple laboratories have implicated Ssy1, a non‐transporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al. 2015 (Traffic 16:135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐exit motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.

  • 22.
    Ring, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Myronidi, Ioanna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The ER membrane chaperone Shr3 co-translationally assists biogenesis of related polytopic membrane protein substratesManuscript (preprint) (Other academic)
    Abstract [en]

    Polytopic membrane proteins with multiple transmembrane segments (TMS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Discrete sets of polytopic membrane proteins in Saccharomyces cerevisiae require ER membrane-localized chaperones (MLC) to prevent aggregation and fold properly. Shr3, the best characterized MLC, is specifically required for the functional expression of amino acid permeases (AAP), a family of transport proteins comprised of twelve TMS. We performed comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with split-ubiquitin approaches to probe chaperone-substrate (Shr3-AAP) interactions in vivo. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions, which initiate after the first 2 to 4 TMS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 TMS are inserted. Thus, Shr3 acts transiently in a co-translationally manner to prevent TMS of translation intermediates from engaging in non-productive interactions, thereby preventing AAP misfolding during biogenesis.

  • 23.
    Silao, Fitz Gerald S.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ward, Meliza
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ryman, Kicki
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wallström, Axel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Brindefalk, Björn
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Udekwu, Klas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans2019In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 2, article id e1007976Article in journal (Refereed)
    Abstract [en]

    Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence. Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.

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