| 1. |
- Boban, Mirta, et al.
(author)
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A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation
- 2014
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:16, s. 3603-3613
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
- Davis, Monica M., et al.
(author)
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Wild-Type Drosophila melanogaster as a Model Host to Analyze Nitrogen Source Dependent Virulence of Candida albicans
- 2011
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:11, s. e27434-
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Journal article (peer-reviewed)abstract
- 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.
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| 3. |
- Khmelinskii, Anton, et al.
(author)
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Protein quality control at the inner nuclear membrane
- 2014
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 516:7531, s. 410-
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Ljungdahl, Per O., et al.
(author)
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Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae
- 2012
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In: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 190:3, s. 885-929
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Omnus, Deike J., 1981-, et al.
(author)
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A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5
- 2011
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 22:15, s. 2754-2765
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Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Omnus, Deike J., et al.
(author)
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Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron
- 2014
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:23, s. 3823-3833
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
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| 8. |
- Omnus, Deike J., 1981-
(author)
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Regulatory mechanisms of amino acid-induced signaling in Saccharomyces cerevisiae
- 2014
-
Doctoral thesis (other academic/artistic)abstract
- This thesis describes studies aimed at elucidating the molecular mechanisms that regulate the SPS (Ssy1-Ptr3-Ssy5) signal transduction pathway in the yeast Saccharomyces cerevisiae. This pathway is induced by extracellular amino acids and facilitates their uptake. The most downstream effectors of the SPS pathway, the homologous transcription factors Stp1 and Stp2 (Stp1/2), are synthesized as latent precursors with N-terminal regulatory domains that restrict their nuclear accumulation. Amino acid-induced signaling, initiated by the plasma membrane localized receptor Ssy1, leads via Ptr3 to the activation of the endoprotease Ssy5. Active Ssy5 cleaves the regulatory domains in Stp1/2. As a consequence, the processed transcription factors lacking their N-terminal domains accumulate in the nucleus and activate the transcription of amino acid permease genes to enhance the uptake capacity of cells.Ssy5 is synthesized as a zymogen precursor that processes itself into a prodomain and catalytic (Cat) domain that remain non-covalently associated. We found that the prodomain functions as an inhibitor of the Cat domain. Signaling triggers the degradation of the prodomain by the proteasome, thereby releasing Cat domain activity (paper I). We identified a motif in the prodomain that functions as inducible phosphodegron. Upon signaling, this motif is phosphorylated which triggers prodomain polyubiquitylation, and as a consequence, its proteasomal degradation (paper II). Also, we found that Ptr3 functions to mediate prodomain phosphorylation upon signaling and that protein phosphatase 2A constitutively mutes phosphorylation-dependent activation of Ssy5 (paper III).Finally, in addition to the regulation of the processing protease Ssy5, the control of transcriptional activity of Stp1 depends on a motif within its N-terminal regulatory domain, designated Region I. We found that Region I mediates latency by functioning as cytoplasmic retention determinant and nuclear degron (paper IV).
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| 9. |
- Omnus, Deike J., 1981-, et al.
(author)
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Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I
- 2013
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 24:9, s. 1480-1492
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Pfirrmann, Thorsten, et al.
(author)
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SOMA : A Single Oligonucleotide Mutagenesis and Cloning Approach
- 2013
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:6
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Journal article (peer-reviewed)abstract
- 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.
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