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Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.
ADAM15 protein amplifies focal adhesion kinase phosphorylation under genotoxic stress conditions
(2012)
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.
Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.
Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown.
Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22.
Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants.
Significance: The findings are important for the understanding of periplasmic protein transport.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
We proposed previously that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane after ethanol exposure leads to suppression of mitochondrial metabolite exchange. Because ureagenesis requires extensive mitochondrial metabolite exchange, we characterized the effect of ethanol and its metabolite, acetaldehyde (AcAld), on total and ureagenic respiration in cultured rat hepatocytes. Ureagenic substrates increased cellular respiration from 15.8 ± 0.9 nmol O(2)/min/10(6) cells (base line) to 29.4 ± 1.7 nmol O(2)/min/10(6) cells in about 30 min. Ethanol (0-200 mM) suppressed extra respiration after ureagenic substrates (ureagenic respiration) by up to 51% but not base line respiration. Urea formation also declined proportionately. Inhibition of alcohol dehydrogenase, cytochrome P450 2E1, and catalase with 4-methylpyrazole, trans-1,2-dichloroethylene, and 3-amino-1,2,3-triazole restored ethanol-suppressed ureagenic respiration by 46, 37, and 66%, respectively. By contrast, inhibition of aldehyde dehydrogenase with phenethyl isothiocyanate increased the inhibitory effect of ethanol on ureagenic respiration by an additional 60%. AcAld, an intermediate product of ethanol oxidation, suppressed ureagenic respiration with an apparent IC(50) of 125 μM. AcAld also inhibited entry of 3-kDa rhodamine-conjugated dextran in the mitochondrial intermembrane space of digitonin-permeabilized hepatocytes, indicative of VDAC closure. In conclusion, AcAld, derived from ethanol metabolism, suppresses ureagenesis in hepatocytes mediated by closure of VDAC.
DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.
Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.
A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.
Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.
The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.
Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX5R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.
Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins.
The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.
Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-μs electron injection into CuA, close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H+/COX and 1 H+/COX, respectively). The subsequent 10-μs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ∼0.5 H+/COX at this side to electrostatically compensate the charge of the electron. With ∼200 μs, all but 0.4 H+ at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called “pump site.” Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to CuA. These results support the idea of a proton-collecting antenna, switched on by electron injection.
Background: The transcription factor T-bet is pivotal for initiation of Th1-related immunoactivation. Identification of novel genes directly regulated by T-bet is crucial.
Results: Genome-wide analysis and subsequent experiments revealed that T-bet up-regulates IL-36γ/IL-1F9 in myeloid cells.
Conclusion: IL-1-related IL-36γ is a direct T-bet target in myeloid cells.
Significance: Observations suggest that IL-36γ , besides IFNγ, contributes to T-bet functions in immunopathology
By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.
Ubiquitin-binding modules are constituents of cellular proteins that mediate the effects of ubiquitylation by making transient, non-covalent interactions with ubiquitin molecules. While some ubiquitin-binding modules bind single ubiquitin moieties, others are selective for specific ubiquitin chains of different linkage types and lengths. In recent years, functions of ubiquitin chains that are polymerized through their Lys or N-terminal Met (i.e. linear chains) residues have been linked to a variety of cellular processes. Selectivity of ubiquitin-binding modules for different ubiquitin chain types appears as a key to the distinct regulatory consequences during protein quality control pathways, receptor endocytosis, gene transcription, signaling via the NF-κB pathway, and autophagy.
The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.
The lichen-forming genus Pertusaria under its current circumscription is polyphyletic and its phylogenetic affiliations are uncertain. Here we study the species of the genera Pertusaria and Varicellaria which containlecanoric acid as major constituent, have disciform apothecia, strongly amyloid asci, non-amyloid hymenial gel, 1-2-spored asci, and 1- or 2-celled ascospores with thick, 1-layered walls. We infer phylogenetic relationships using maximum likelihood and Bayesian analyses based on four molecular loci (mtSSU, nuLSU rDNA, and the protein-coding, nuclear RPB1 and MCM7 genes). Our results show that the lecanoric acid-containing species form a well-supported, monophyletic group, which is only distantly related to Pertusaria s.str. The phylogenetic position of this clade is unclear, but placement in Pertusaria s.str. is rejected using alternative hypothesis testing. The circumscription of the genus Varicellaria is enlarged to also include species with non-septate ascospores. Seven species are accepted in the genus: Varicellaria culbersonii (Vězda) Schmitt & Lumbsch, comb. nov., Varicellaria hemisphaerica (Flörke) Schmitt & Lumbsch, comb. nov., Varicellaria kasandjeffii (Szatala) Schmitt & Lumbsch, comb. nov., Varicellaria lactea (L.) Schmitt & Lumbsch, comb. nov., Varicellaria philippina (Vain.) Schmitt & Lumbsch, comb. nov., Varicellaria rhodocarpa (Körb.) Th. Fr., and Varicellaria velata (Turner) Schmitt & Lumbsch, comb. nov. A key to the species of Varicellaria is provided.