Publications by authors named "Kerstin Schmitt"

19 Publications

  • Page 1 of 1

EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain.

Front Plant Sci 2021 14;12:658961. Epub 2021 Apr 14.

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein's C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of gene expression in revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.
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http://dx.doi.org/10.3389/fpls.2021.658961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079945PMC
April 2021

The velvet protein Vel1 controls initial plant root colonization and conidia formation for xylem distribution in Verticillium wilt.

PLoS Genet 2021 Mar 15;17(3):e1009434. Epub 2021 Mar 15.

Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, University of Göttingen and Göttingen Center for Molecular Biosciences (GZMB), Göttingen, Germany.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.
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http://dx.doi.org/10.1371/journal.pgen.1009434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993770PMC
March 2021

Crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP.

Acta Crystallogr F Struct Biol Commun 2021 Mar 3;77(Pt 3):70-78. Epub 2021 Mar 3.

Department for Molecular Structural Biology, Georg-August-Universität Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany.

CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58 Å resolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.
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http://dx.doi.org/10.1107/S2053230X2100203XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938638PMC
March 2021

A meet-up of two second messengers: the c-di-AMP receptor DarB controls (p)ppGpp synthesis in Bacillus subtilis.

Nat Commun 2021 02 22;12(1):1210. Epub 2021 Feb 22.

Department of General Microbiology, Institute for Microbiology & Genetics, GZMB, Georg-August-University Göttingen, Göttingen, Germany.

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB-Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
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http://dx.doi.org/10.1038/s41467-021-21306-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900238PMC
February 2021

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi.

PLoS Genet 2020 08 25;16(8):e1008996. Epub 2020 Aug 25.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Bloco Q, Universidade de São Paulo, Brazil.

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.
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http://dx.doi.org/10.1371/journal.pgen.1008996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473523PMC
August 2020

The Novel J-Domain Protein Mrj1 Is Required for Mitochondrial Respiration and Virulence in Cryptococcus neoformans.

mBio 2020 06 9;11(3). Epub 2020 Jun 9.

Michael Smith Laboratories, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

The opportunistic fungal pathogen must adapt to the mammalian environment to establish an infection. Proteins facilitating adaptation to novel environments, such as chaperones, may be required for virulence. In this study, we identified a novel mitochondrial co-chaperone, Mrj1 (itochondrial espiration -domain protein ), necessary for virulence in The and J-domain-inactivated mutants had general growth defects at both routine laboratory and human body temperatures and were deficient in the major virulence factor of capsule elaboration. The latter phenotype was associated with cell wall changes and increased capsular polysaccharide shedding. Accordingly, the mutant was avirulent in a murine model of cryptococcosis. Mrj1 has a mitochondrial localization and co-immunoprecipitated with Qcr2, a core component of complex III of the electron transport chain. The mutants were deficient in mitochondrial functions, including growth on alternative carbon sources, growth without iron, and mitochondrial polarization. They were also insensitive to complex III inhibitors and hypersensitive to an alternative oxidase (AOX) inhibitor, suggesting that Mrj1 functions in respiration. In support of this conclusion, mutants also had elevated basal oxygen consumption rates which were completely abolished by the addition of the AOX inhibitor, confirming that Mrj1 is required for mitochondrial respiration through complexes III and IV. Furthermore, inhibition of complex III phenocopied the capsule and cell wall defects of the mutants. Taken together, these results indicate that Mrj1 is required for normal mitochondrial respiration, a key aspect of adaptation to the host environment and virulence. is the causative agent of cryptococcal meningitis, a disease responsible for ∼15% of all HIV-related deaths. Unfortunately, development of antifungal drugs is challenging because potential targets are conserved between humans and In this context, we characterized a unique J-domain protein, Mrj1, which lacks orthologs in humans. We showed that Mrj1 was required for normal mitochondrial respiration and that mutants lacking Mrj1 were deficient in growth, capsule elaboration, and virulence. Furthermore, we were able to phenocopy the defects in growth and capsule elaboration by inhibiting respiration. This result suggests that the role of Mrj1 in mitochondrial function was responsible for the observed virulence defects and reinforces the importance of mitochondria to fungal pathogenesis. Mitochondria are difficult to target, as their function is also key to human cells; however, Mrj1 presents an opportunity to target a unique fungal protein required for mitochondrial function and virulence in .
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http://dx.doi.org/10.1128/mBio.01127-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373193PMC
June 2020

Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings.

Plant Physiol 2020 03 11;182(3):1326-1345. Epub 2019 Dec 11.

University of Göttingen, Albrecht-von-Haller-Institute for Plant Sciences and Göttingen Center for Molecular Biosciences (GZMB), Department of Plant Biochemistry, 37077 Göttingen, Germany

The developmental program of seed formation, germination, and early seedling growth requires not only tight regulation of cell division and metabolism, but also concerted control of the structure and function of organelles, which relies on specific changes in their protein composition. Of particular interest is the switch from heterotrophic to photoautotrophic seedling growth, for which cytoplasmic lipid droplets (LDs) play a critical role as depots for energy-rich storage lipids. Here, we present the results of a bottom-up proteomics study analyzing the total protein fractions and LD-enriched fractions in eight different developmental phases during silique (seed) development, seed germination, and seedling establishment in Arabidopsis (). The quantitative analysis of the LD proteome using LD-enrichment factors led to the identification of six previously unidentified and comparably low-abundance LD proteins, each of which was confirmed by intracellular localization studies with fluorescent protein fusions. In addition to these advances in LD protein discovery and the potential insights provided to as yet unexplored aspects in plant LD functions, our data set allowed for a comparative analysis of the LD protein composition throughout the various developmental phases examined. Among the most notable of the alterations in the LD proteome were those during seedling establishment, indicating a switch in the physiological function(s) of LDs after greening of the cotyledons. This work highlights LDs as dynamic organelles with functions beyond lipid storage.
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http://dx.doi.org/10.1104/pp.19.01255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054876PMC
March 2020

yRACK1/Asc1 proxiOMICs-Towards Illuminating Ships Passing in the Night.

Cells 2019 11 4;8(11). Epub 2019 Nov 4.

Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany.

Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit () and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the . We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.
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http://dx.doi.org/10.3390/cells8111384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912217PMC
November 2019

Mapping Cellular Microenvironments: Proximity Labeling and Complexome Profiling (Seventh Symposium of the Göttingen Proteomics Forum).

Cells 2019 10 2;8(10). Epub 2019 Oct 2.

Institute of Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany.

Mass spectrometry-based proteomics methods are finding increasing use in structural biology research. Beyond simple interaction networks, information about stable protein-protein complexes or spatially proximal proteins helps to elucidate the biological functions of proteins in a wider cellular context. To shed light on new developments in this field, the Göttingen Proteomics Forum organized a one-day symposium focused on complexome profiling and proximity labeling, two emerging technologies that are gaining significant attention in biomolecular research. The symposium was held in Göttingen, Germany on 23 May, 2019, as part of a series of regular symposia organized by the Göttingen Proteomics Forum.
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http://dx.doi.org/10.3390/cells8101192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830108PMC
October 2019

COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development.

Biomolecules 2019 06 18;9(6). Epub 2019 Jun 18.

Department of Molecular Microbiology and Genetics and Goettingen Center for Molecular Biosciences (GZMB), University of Goettingen, 37077 Goettingen, Germany.

COP9 signalosome (CSN) and Den1/A deneddylases physically interact and promote multicellular development in fungi. CSN recognizes Skp1/cullin-1/Fbx E3 cullin-RING ligases (CRLs) without substrate and removes their posttranslational Nedd8 modification from the cullin scaffold. This results in CRL complex disassembly and allows Skp1 adaptor/Fbx receptor exchange for altered substrate specificity. We characterized the novel ubiquitin-specific protease UspA of the mold , which corresponds to CSN-associated human Usp15 and interacts with six CSN subunits. UspA reduces amounts of ubiquitinated proteins during fungal development, and the gene expression is repressed by an intact CSN. UspA is localized in proximity to nuclei and recruits proteins related to nuclear transport and transcriptional processing, suggesting functions in nuclear entry control. UspA accelerates the formation of asexual conidiospores, sexual development, and supports the repression of secondary metabolite clusters as the () genes. UspA reduces protein levels of the fungal NF-kappa B-like velvet domain protein VeA, which coordinates differentiation and secondary metabolism. VeA stability depends on the Fbx23 receptor, which is required for light controlled development. Our data suggest that the interplay between CSN deneddylase, UspA deubiquitinase, and SCF-Fbx23 ensures accurate levels of VeA to support fungal development and an appropriate secondary metabolism.
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http://dx.doi.org/10.3390/biom9060238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627422PMC
June 2019

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins.

Plant Cell 2018 09 7;30(9):2137-2160. Epub 2018 Aug 7.

Department of Plant Biochemistry, Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, 37077 Göttingen, Germany

The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco () pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in and expressed in embryos, pollen tubes, and seedlings. In knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.
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http://dx.doi.org/10.1105/tpc.18.00276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181012PMC
September 2018

Integrative omics - from data to biology.

Expert Rev Proteomics 2018 06 18;15(6):463-466. Epub 2018 May 18.

a Göttingen Proteomics Forum (GPF) , Göttingen , Germany.

Introduction: Multi-omic approaches are promising a broader view on cellular processes and a deeper understanding of biological systems. with strongly improved high-throughput methods the amounts of data generated have become huge, and their handling challenging. Area Covered: New bioinformatic tools and pipelines for the integration of data from different omics disciplines continue to emerge, and will support scientists to reliably interpret data in the context of biological processes. comprehensive data integration strategies will fundamentally improve systems biology and systems medicine. to present recent developments of integrative omics, the göttingen proteomics forum (gpf) organized its 6th symposium on the 23rd of november 2017, as part of a series of regular gpf symposia. more than 140 scientists attended the event that highlighted the challenges and opportunities but also the caveats of integrating data from different omics disciplines. Expert commentary: The continuous exponential growth in omics data require similar development in software solutions for handling this challenge. Integrative omics tools offer the chance to handle this challenge but profound investigations and coordinated efforts are required to boost this field.
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http://dx.doi.org/10.1080/14789450.2018.1476143DOI Listing
June 2018

Extensive Identification and In-depth Validation of Importin 13 Cargoes.

Mol Cell Proteomics 2018 07 17;17(7):1337-1353. Epub 2018 Apr 17.

From the ‡Department of Molecular Biology, Faculty of Medicine, Göttingen Center of Biosciences (GZMB), Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany;

Importin 13 is a member of the importin β family of transport receptors. Unlike most family members, importin 13 mediates both, nuclear protein import and export. To search for novel importin 13 cargoes, we used stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Using stringent criteria, we identified 255 importin 13 substrates, including the known cargoes Ubc9, Mago and eIF1A, and validate many of them as transport cargoes by extensive biochemical and cell biological characterization. Several novel cargoes can also be transported by the export receptor CRM1, demonstrating a clear redundancy in receptor choice. Using importin 13 mutants, we show that many of the novel substrates contact regions on the transport receptor that are not used by Ubc9, Mago or eIF1A. Together, this study significantly expands the repertoire of importin 13 cargoes and sets the basis for a more detailed characterization of this extremely versatile transport receptor.
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http://dx.doi.org/10.1074/mcp.RA118.000623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030721PMC
July 2018

Capturing the Asc1p/eceptor for ctivated inase (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast.

Mol Cell Proteomics 2017 Dec 5;16(12):2199-2218. Epub 2017 Oct 5.

From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany;

The Asc1 protein of is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent tin entification (BioID), an labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1p, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins ( of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes.
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http://dx.doi.org/10.1074/mcp.M116.066654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724181PMC
December 2017

Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling.

Mol Cell Biol 2017 02 19;37(3). Epub 2017 Jan 19.

Department of Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Georg-August University, Göttingen, Germany

WD40 repeat proteins fold into characteristic β-propeller structures and control signaling circuits during cellular adaptation processes within eukaryotes. The RACK1 protein of Saccharomyces cerevisiae, Asc1p, consists exclusively of a single seven-bladed β-propeller that operates from the ribosomal base at the head region of the 40S subunit. Here we show that the R38D K40E ribosomal binding-compromised variant (Asc1DEp) is severely destabilized through mutation of phosphosite T143 to a dephosphorylation-mimicking alanine, probably through proteasomal degradation, leading to asc1 phenotypes. Phosphosite Y250 contributes to resistance to translational inhibitors but does not influence Asc1DEp stability. Beyond its own phosphorylation at T143, Y250, and other sites, Asc1p heavily influences the phosphorylation of as many as 90 proteins at 120 sites. Many of these proteins are regulators of fundamental processes ranging from mRNA translation to protein transport and turnover, cytoskeleton organization, and cellular signaling. Our data expose Asc1p/RACK1 as a key factor in phosphosignaling and manifest it as a control point at the head of the ribosomal 40S subunit itself regulated through posttranslational modification.
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http://dx.doi.org/10.1128/MCB.00279-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247610PMC
February 2017

Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1.

PLoS Genet 2014 Apr 24;10(4):e1004306. Epub 2014 Apr 24.

Institute for Biology II - Molecular Plant Physiology, Albert-Ludwigs University Freiburg, Freiburg, Germany; Freiburg Institute for Advanced Studies (FRIAS), Albert-Ludwigs University Freiburg, Freiburg, Germany.

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.
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http://dx.doi.org/10.1371/journal.pgen.1004306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998894PMC
April 2014

Phospho-regulation of the Neurospora crassa septation initiation network.

PLoS One 2013 21;8(10):e79464. Epub 2013 Oct 21.

Institute for Biology II - Molecular Plant Physiology, Albert-Ludwigs University Freiburg, Freiburg, Germany.

Proper cell division is essential for growth and development of uni- and multicellular organisms. The fungal septation initiation network (SIN) functions as kinase cascade that connects cell cycle progression with the initiation of cytokinesis. Miss-regulation of the homologous Hippo pathway in animals results in excessive cell proliferation and formation of tumors, underscoring the conservation of both pathways. How SIN proteins interact and transmit signals through the cascade is only beginning to be understood. Moreover, our understanding of septum formation and its regulation in filamentous fungi, which represent the vast majority of the fungal kingdom, is highly fragmentary. We determined that a tripartite kinase cascade, consisting of CDC-7, SID-1 and DBF-2, together with their regulatory subunits CDC-14 and MOB-1, is important for septum formation in the model mold Neurospora crassa. DBF-2 activity and septum formation requires auto-phosphorylation at Ser499 within the activation segment and phosphorylation of Thr671 in the hydrophobic motif by SID-1. Moreover, SID-1-stimulated DBF-2 activity is further enhanced by CDC-7, supporting a stepwise activation mechanism of the tripartite SIN kinase cascade in fungi. However, in contrast to the situation described for unicellular yeasts, the localization of the entire SIN cascade to spindle pole bodies is constitutive and cell cycle independent. Moreover, all SIN proteins except CDC-7 form cortical rings prior to septum initiation and localize to constricting septa. Thus, SIN localization and activity regulation significantly differs in unicellular versus syncytial ascomycete fungi.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804505PMC
February 2015

HAM-2 and HAM-3 are central for the assembly of the Neurospora STRIPAK complex at the nuclear envelope and regulate nuclear accumulation of the MAP kinase MAK-1 in a MAK-2-dependent manner.

Mol Microbiol 2013 Nov 1;90(4):796-812. Epub 2013 Oct 1.

Institute for Biology II - Molecular Plant Physiology, Albert-Ludwigs University Freiburg, Schänzlestr. 1, 79104, Freiburg, Germany.

Intercellular communication and somatic cell fusion are important for fungal colony establishment, multicellular differentiation and have been associated with host colonization and virulence of pathogenic species. By a combination of genetic, biochemical and live cell imaging techniques, we characterized the Neurospora crassa STRIPAK complex that is essential for self-signalling and consists of the six proteins HAM-2/STRIP, HAM-3/striatin, HAM-4/SLMAP, MOB-3/phocein, PPG-1/PP2A-C and PP2A-A. We describe that the core STRIPAK components HAM-2 and HAM-3 are central for the assembly of the complex at the nuclear envelope, while the phosphatase PPG-1 only transiently associates with this central subcomplex. Our data connect the STRIPAK complex with two MAP kinase pathways: (i) nuclear accumulation of the cell wall integrity MAP kinase MAK-1 depends on the functional integrity of the STRIPAK complex at the nuclear envelope, and (ii) phosphorylation of MOB-3 by the MAP kinase MAK-2 impacts the nuclear accumulation of MAK-1. In summary, these data support a model, in which MAK-2-dependent phosphorylation of MOB-3 is part of a MAK-1 import mechanism. Although self-communication remained intact in the absence of nuclear MAK-1 accumulation, supporting the presence of multiple mechanisms that co-ordinate robust intercellular communication, proper fruiting body morphology was dependent on the MAK-2-phosphorylated N-terminus of MOB-3.
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http://dx.doi.org/10.1111/mmi.12399DOI Listing
November 2013

RACK1/Asc1p, a ribosomal node in cellular signaling.

Mol Cell Proteomics 2013 Jan 15;12(1):87-105. Epub 2012 Oct 15.

Institute of Microbiology and Genetics, Georg-August Universität, D-37077 Göttingen, Germany.

RACK1/Asc1p and its essential orthologues in higher eukaryotes, such as RACK1 in metazoa, are involved in several distinct cellular signaling processes. The implications of a total deletion have never been assessed in a comprehensive manner. This study reveals the major cellular processes affected in a Saccharomyces cerevisiae Δasc1 deletion background via de novo proteome and transcriptome analysis, as well as subsequent phenotypical characterizations. The deletion of ASC1 reduces iron uptake and causes nitrosative stress, both known indicators for hypoxia, which manifests in a shift of energy metabolism from respiration to fermentation in the Δasc1 strain. Asc1p further impacts cellular metabolism through its regulative role in the MAP kinase signal transduction pathways of invasive/filamentous growth and cell wall integrity. In the Δasc1 mutant strain, aberrations from the expected cellular response, mediated by these pathways, can be observed and are linked to changes in protein abundances of pathway-targeted transcription factors. Evidence of the translational regulation of such transcription factors suggests that ribosomal Asc1p is involved in signal transduction pathways and controls the biosynthesis of the respective final transcriptional regulators.
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http://dx.doi.org/10.1074/mcp.M112.017277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536911PMC
January 2013