Fungal COP9 signalosome assembly requires connection of two trimeric intermediates for integration of intrinsic deneddylase.

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

Verticillium dahliae Vta3 promotes ELV1 virulence factor gene expression in xylem sap, but tames Mtf1-mediated late stages of fungus-plant interactions and microsclerotia formation.

Verticillium transcription activator of adhesion 3 (Vta3) is required for plant root colonization and pathogenicity of the soil-borne vascular fungus Verticillium dahliae. RNA sequencing identified Vta3-dependent genetic networks required for growth in tomato xylem sap. Vta3 affects the expression of more than 1,000 transcripts, including candidates with predicted functions in virulence and morphogenesis such as Egh16-like virulence factor 1 (Elv1) and Master transcription factor 1 (Mtf1). The genes encoding Elv1 and Mtf1 were deleted and their functions in V. dahliae growth and virulence on tomato (Solanum lycopersicum) plants were investigated using genetics, plant infection experiments, gene expression studies and phytohormone analyses. Vta3 contributes to virulence by promoting ELV1 expression, which is dispensable for vegetative growth and conidiation. Vta3 decreases disease symptoms mediated by Mtf1 in advanced stages of tomato plant colonization, while Mtf1 induces the expression of fungal effector genes and tomato pathogenesis-related protein genes. The levels of pipecolic and salicylic acids functioning in tomato defense signaling against (hemi-) biotrophic pathogens depend on the presence of MTF1, which promotes the formation of resting structures at the end of the infection cycle. In summary, the presence of VTA3 alters gene expression of virulence factors and tames the Mtf1 genetic subnetwork for late stages of plant disease progression and subsequent survival of the fungus in the soil.

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress.

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis (Arabidopsis thaliana) leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant trigalactosyldiacylglycerol1-1 sugar dependent 1-4 (tgd1-1 sdp1-4) that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we showed that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress.

SEED LIPID DROPLET PROTEIN1, SEED LIPID DROPLET PROTEIN2, and LIPID DROPLET PLASMA MEMBRANE ADAPTOR mediate lipid droplet-plasma membrane tethering.

Membrane contact sites (MCSs) are interorganellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here, we identified and characterized three proteins of Arabidopsis thaliana that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localized SEED LD PROTEIN (SLDP) 1 and SLDP2 and PM-localized LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and SLDP2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterized by dynamically moving LDs in the cytosolic streaming. Furthermore, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis.

F-box receptor mediated control of substrate stability and subcellular location organizes cellular development of Aspergillus nidulans.

Fungal growth and development are coordinated with specific secondary metabolism. This coordination requires 8 of 74 F-box proteins of the filamentous fungus Aspergillus nidulans. F-box proteins recognize primed substrates for ubiquitination by Skp1-Cul1-Fbx (SCF) E3 ubiquitin RING ligases and degradation by the 26S proteasome. 24 F-box proteins are found in the nuclear fraction as part of SCFs during vegetative growth. 43 F-box proteins interact with SCF proteins during growth, development or stress. 45 F-box proteins are associated with more than 700 proteins that have mainly regulatory roles. This corroborates that accurate surveillance of protein stability is prerequisite for organizing multicellular fungal development. Fbx23 combines subcellular location and protein stability control, illustrating the complexity of F-box mediated regulation during fungal development. Fbx23 interacts with epigenetic methyltransferase VipC which interacts with fungal NF-κB-like velvet domain regulator VeA that coordinates fungal development with secondary metabolism. Fbx23 prevents nuclear accumulation of methyltransferase VipC during early development. These results suggest that in addition to their role in protein degradation, F-box proteins also control subcellular accumulations of key regulatory proteins for fungal development.

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

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.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate.

Xylem sap is the major transport route for nutrients from roots to shoots. In the present study, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap and the growth of the xylem endophyte Brennaria salicis, and we also report transcriptional re-wiring of leaf defenses in poplar (Populus × canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. Approximately 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, whereas nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased, whereas salicylic acid and jasmonoyl-isoleucine decreased, with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 124 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N, except coumarins. Brennaria salicis growth was reduced in nutrient-supplemented xylem sap of low- and high- NO3- -fed plants compared to that of NH4+ -fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defenses related to systemic acquired and induced systemic resistance. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

The Nma1 protein promotes long distance transport mediated by early endosomes in Ustilago maydis.

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.

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

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.

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

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 (Arabidopsis thaliana). 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.

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

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 (Nicotiana tabacum) 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 Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 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.

A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the ß-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus.

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

Importin 13 is a member of the importin ß 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.

Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast.

The Asc1 protein of Saccharomyces cerevisiae 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 Biotin IDentification (BioID), an in vivo 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 Asc1R38D, K40Ep, 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 ß-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (e.g. of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes.

Integrative omics - from data to biology.

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.

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

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.

RACK1/Asc1p, a ribosomal node in cellular signaling.

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.

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.

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.