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.

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.

The Frq-Frh Complex Light-Dependently Delays Sfl1-Induced Microsclerotia Formation in Verticillium dahliae.

The vascular plant pathogenic fungus Verticillium dahliae has to adapt to environmental changes outside and inside its host. V. dahliae harbors homologs of Neurospora crassa clock genes. The molecular functions and interactions of Frequency (Frq) and Frq-interacting RNA helicase (Frh) in controlling conidia or microsclerotia development were investigated in V. dahliae JR2. Fungal mutant strains carrying clock gene deletions, an FRH point mutation, or GFP gene fusions were analyzed on transcript, protein, and phenotypic levels as well as in pathogenicity assays on tomato plants. Our results support that the Frq-Frh complex is formed and that it promotes conidiation, but also that it suppresses and therefore delays V. dahliae microsclerotia formation in response to light. We investigated a possible link between the negative element Frq and positive regulator Suppressor of flocculation 1 (Sfl1) in microsclerotia formation to elucidate the regulatory molecular mechanism. Both Frq and Sfl1 are mainly present during the onset of microsclerotia formation with decreasing protein levels during further development. Induction of microsclerotia formation requires Sfl1 and can be delayed at early time points in the light through the Frq-Frh complex. Gaining further molecular knowledge on V. dahliae development will improve control of fungal growth and Verticillium wilt disease.

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.

Vacuole fragmentation depends on a novel Atg18-containing retromer-complex.

The yeast PROPPIN Atg18 folds as a ß-propeller with two binding sites for phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,5)P2 at its circumference. Membrane insertion of an amphipathic loop of Atg18 leads to membrane tubulation and fission. Atg18 has known functions at the PAS during macroautophagy, but the functional relevance of its endosomal and vacuolar pool is not well understood. Here we show in a proximity-dependent labeling approach and by co-immunoprecipitations that Atg18 interacts with Vps35, a central component of the retromer complex. The binding of Atg18 to Vps35 is competitive with the sorting nexin dimer Vps5 and Vps17. This suggests that Atg18 within the retromer can substitute for both the phosphoinositide binding and the membrane bending capabilities of these sorting nexins. Indeed, we found that Atg18-retromer is required for PtdIns(3,5)P2-dependent vacuolar fragmentation during hyperosmotic stress. The Atg18-retromer is further involved in the normal sorting of the integral membrane protein Atg9. However, PtdIns3P-dependent macroautophagy and the selective cytoplasm-to-vacuole targeting (Cvt) pathway are only partially affected by the Atg18-retromer. We expect that this is due to the plasticity of the different sorting pathways within the endovacuolar system.Abbreviations: BAR: bin/amphiphysin/Rvs; FOA: 5-fluoroorotic acid; PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology.

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.

Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora.

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC-MS). In contrast to many other affinity capture approaches for studying protein-protein interactions, BioID does not rely on physical protein-protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordaria macrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S. macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S. macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

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.

Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells.

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

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.

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.

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.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen.

Histone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a dnj4Δ deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the dnj4Δ mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the dnj4Δ mutant. Accordingly, excess iron rescued the mutant's growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans. IMPORTANCE DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones. In this study, we show that a histone chaperone, Dnj4, is required for genome integrity and for the response to DNA damage. The gene encoding this protein in Cryptococcus neoformans lacks an ortholog in Saccharomyces cerevisiae; however, it is conserved in humans in which its ortholog is essential. Since it is not essential in C. neoformans, we were able to generate deletion mutants to characterize the roles of Dnj4. We also expressed Dnj4 in S. cerevisiae, in which it was able to bind S. cerevisiae histones and interfere with existing histone chaperoning machinery. Therefore, we show a conserved role for Dnj4 in histone chaperoning that suggests that C. neoformans is useful to better understand aspects of this important biological process.

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.

Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora.

In the filamentous fungus Sordaria macrospora (Sm), the STRIPAK complex is required for vegetative growth, fruiting-body development and hyphal fusion. The SmSTRIPAK core consists of the striatin homolog PRO11, the scaffolding subunit of phosphatase PP2A, SmPP2AA, and its catalytic subunit SmPP2Ac1. Among other STRIPAK proteins, the recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Pulldown experiments with SCI identified the transmembrane nucleoporin (TM Nup) SmPOM33 as a potential nuclear-anchor of SmSTRIPAK. Localization studies revealed that SmPOM33 partially localizes to the nuclear envelope (NE), but mainly to the endoplasmic reticulum (ER). We succeeded to generate a Δpom33 deletion mutant by homologous recombination in a new S. macrospora Δku80 recipient strain, which is defective in non-homologous end joining. Deletion of Smpom33 did neither impair vegetative growth nor sexual development. In pulldown experiments of SmPOM33 followed by LC/MS analysis, ER-membrane proteins involved in ER morphology, protein translocation, glycosylation, sterol biosynthesis and Ca2+-transport were significantly enriched. Data are available via ProteomeXchange with identifier PXD026253. Although no SmSTRIPAK components were identified as putative interaction partners, it cannot be excluded that SmPOM33 is involved in temporarily anchoring the SmSTRIPAK to the NE or other sites in the cell.

α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.

Parkinson's disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

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

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 Arabidopsis 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 Arabidopsis 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 ERD7 gene expression in Arabidopsis 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.

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

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 Šresolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.

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.

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

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.