The Cell End Marker Protein TeaC Is Involved in Growth Directionality and Septation in Aspergillus nidulans

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In Schizosaccharomyces pombe it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to Aspergillus nidulans. Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of teaC affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of teaA deletion (tea1) strains. Some small, anucleate compartments were observed. Overexpression of teaC repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.Filamentous fungi represent fascinating model organisms for studying the establishment and maintenance of cell polarity, because cell growth takes place at the tip of the extremely elongated hyphae. Hyphal extension requires the continuous expansion of the membrane and the cell wall and is driven by continuous fusion of secretion vesicles at the tip (8, 12). The transportation of vesicles is probably achieved by the coordinated action of the MT and the actin cytoskeleton. According to one model, vesicles first travel along MTs, are unloaded close to the hyphal tip, where they form a microscopically visible structure the “Spitzenkörper,” which is also called the “vesicle supply center,” referring to the assumed function (24, 25). For the last step, vesicle transportation from the Spitzenkörper to the apical membrane, actin-myosin-dependent movement is used. Anti-cytoskeletal drug experiments have shown that hyphae can grow for some time in the absence of MTs but not in the absence of the actin cytoskeleton (14, 27, 30a).In Schizosaccharomyces pombe it was shown clearly that the polarization of the actin cytoskeleton depends on the MT cytoskeleton (2, 7). In 1994, polarity mutants of S. pombe were isolated and subsequent cloning of one of the genes identified the polarity determinant Tea1 (19, 29). Because this protein labels the growing cell end, this and other subsequently isolated proteins of this class were named cell end markers. It was shown that cell end localization of Tea1 requires the activity of a kinesin motor protein, Tea2, which transports the protein to the MT plus end (3). Together with the growing MT, Tea1 reaches the cortex, where it is unloaded and binds to a prenylated and membrane-anchored receptor protein, Mod5 (28). The formin For3, which catalyzes actin cable formation, is recruited to the tip through binding to another cell end marker protein, Tea4, which confers tethering to Tea1 (7, 18, 33). Tea4 is required for For3 localization at the cell tip, specifically during initiation of bipolar growth (18).Recently, it was shown that components of this polarity determination machinery are conserved in the filamentous fungus A. nidulans (8). The first component identified was the Tea2 homologue, KipA, a kinesin-7 motor protein (16). Deletion of the gene did not affect hyphal tip extension but polarity determination. Instead of growing straight, hyphae grew in curves. KipA moves along MTs and accumulates at the MT plus end. The identification of Tea1 and a Mod5 homologue was more difficult, because the primary structure of these cell end marker proteins is not well conserved in filamentous fungi. A Tea1 homologue, TeaA, only displayed 27% sequence identity. However, the presence of Kelch repeats in both proteins suggested conserved functions (31). A Mod5 homologue was identified by a conserved CAAX prenylation motif at the C terminus. Systematic analyses of proteins with such a motif in the A. nidulans genome led to the identification of TeaR. Like Tea1 and Mod5, TeaA and TeaR localize at or close to the hyphal membrane at the growing cell end (31). However, correct localization of TeaR requires TeaA. In addition, sterol-rich membrane domains define the place of TeaR attachment to the hyphal tip. In contrast to S. pombe, TeaA and TeaR are still transported to the hyphal tip in the absence of the motor protein KipA, but their localization is disturbed in comparison to wild type. This suggests that other proteins are necessary for exact TeaA positioning, whose localization depends on KipA.We characterized a homologue of the S. pombe cell end marker protein, Tea4, and found that the protein is required for the maintenance of straight polar growth but that it also appears to be involved in septation.     

Mitochondrial beta-oxidation in Aspergillus nidulans

Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox. While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids. In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids. Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.     

拟南芥钙依赖蛋白激酶参与植物激素信号转导

在植物信号通路中,涉及到钙应答的蛋白激酶大多是钙依赖蛋白激酶。钙依赖蛋白激酶作为钙信号转导因子,参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥AtCPK30基因的功能进行了深入的研究。RT-PCR分析结果表明:AtCPK30在植物根中的表达量很高,其在幼苗中的转录水平分别受ABA、IAA、2,4-D、GA_3和6-BA等激素的诱导调节。AtCPK30基因过表达的转基因株系幼苗的主根比野生型的长,同时发现转基因植株幼苗的根在缺钙的MS培养基上生长较野生型植株长,表明缺钙对转基因幼苗影响较小。用ABA、IAA、GA_3和BA处理时,转基因植株幼苗的根对激素更敏感。当野生型和转基因植株生长在含有生长素抑制剂NPA的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。GFP-CPK30融合蛋白的亚细胞定位研究结果表明:CPK30蛋白定位在细胞壁和细胞膜上。这些研究结果说明了AtCPK30作为钙信号转导因子,参与了多种激素调节植物根生长的过程。     

Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.Protein glycosylation, which is a major posttranslational modification, plays essential roles in eukaryotic cells from fungi to mammals (19). N-linked oligosaccharides in glycoproteins that share relatively common structures are structurally classified into high-mannose, complex, and hybrid types (3). O-linked oligosaccharides in glycoproteins are diverse with respect to their sugar components and the mode of sugar linkages among the eukaryotic organisms (8, 19). O mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (4, 21, 35). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalyzed by protein O-d-mannosyltransferase (Pmt) in the endoplasmic reticulum (ER), where dolichyl phosphate-mannose is required as an immediate sugar donor (4). In the Golgi complex, O mannosylation in S. cerevisiae is linearly elongated by up to five mannose residues by mannosyltransferases (Mnt) that utilize GDP-mannose as the mannosyl donor. At least six Pmt-encoding genes (PMT1 to -6), three α-1,2-Mnt-encoding genes (KRE2, KTR1, and KTR3), and three α-1,3-Mnt-encoding genes (MNN1, MNT2, and MNT3) are known to be involved in O mannosylation in S. cerevisiae (21, 31, 45).The Pmt family of proteins can be classified into the PMT1, PMT2, and PMT4 subfamilies based on phylogeny (6). Proteins of the PMT1 subfamily form a heteromeric complex with proteins belonging to the PMT2 subfamily, and PMT4 subfamily proteins form a homomeric complex (7). Simultaneous disruptions of three different types of PMT genes were lethal (4), suggesting that each class provided a unique function for O mannosylation. Yeasts other than S. cerevisiae, such as Schizosaccharomyces pombe (38, 41), Candida albicans (29), and Cryptococcus neoformans (28), possess three to five pmt genes, which have been characterized. Several studies provide evidence that protein O mannosylation modulates the functions and stability of secretory proteins and thereby affects the growth and morphology of these yeasts. O mannosylation by Pmt2 in S. cerevisiae (ScPmt2) provides protection from ER-associated degradation and also functions as a fail-safe mechanism for ER-associated degradation (11, 13, 23). Likewise, in C. albicans, CaPmt1- and CaPmt4-mediated O mannosylation specifically protects CaSec20 from proteolytic degradation in the ER (40). Cell wall integrity is maintained in S. cerevisiae by increased stabilization and correct localization of the sensor proteins ScWsc and ScMid2 due to O mannosylation by ScPmt2 and ScPmt4 (20). Similarly, the stability and localization to the plasma membrane of axial budding factor ScAxl2/Bud10 is enhanced by ScPmt4-mediated O mannosylation, increasing its activity (32). ScPmt4-mediated O glycosylation also functions as a sorting determinant for cell surface delivery of ScFus1 (30). CaPmt4-mediated O glycosylation is required for environment-specific morphogenetic signaling and for the full virulence of C. albicans (29).With respect to filamentous fungi like Aspergillus that develop hyphae in a highly ordered manner, which then differentiate to form conidiospores, little is known about the function and synthetic pathway of the O-mannose-type oligosaccharides. O-Glycans in glycoproteins of Aspergillus include sugars other than mannose, and their structures have been determined (8). The initial mannosylation catalyzed by Pmts is found in Aspergillus and occurs as in yeasts (8).We characterized the pmtA gene of Aspergillus nidulans (AnpmtA), belonging to the PMT2 subfamily, and found that the mutant exhibited a fragile cell wall phenotype and alteration in the carbohydrate composition, with a reduction in the amount of skeletal polysaccharides in the cell wall (26, 33). Recently, the Afpmt1 gene belonging to the PMT1 family of Aspergillus fumigatus, a human pathogen, was characterized. AfPmt1 is crucial for cell wall integrity and conidium morphology (46).In this study, we characterize the pmtB and pmtC genes of A. nidulans to understand their contribution to the cell morphology of this filamentous fungus. We also demonstrate that the PmtA, PmtB, and PmtC proteins have distinct specificities for protein substrates and function differently during cell growth of filamentous fungi.     

Involvement of Protein Kinase C in the Suppression of Apoptosis and in Polarity Establishment in Aspergillus nidulans under Conditions of Heat Stress

The pkcA gene, which encodes a protein kinase C (PKC) in the filamentous fungus Aspergillus nidulans, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of pkcA. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of bckA, an ortholog of Saccharomyces cerevisiae bck1 that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the bckA deletion mutant under conditions of heat stress, was not observed in the pkcA-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions.     

蛋白激酶Cα相互作用蛋白的结构与功能

蛋白激酶Cα相互作用蛋白（protein interacting with Cα kinase,PICK1）是蛋白激酶Cox（protein kinase Cα,PKCα）的靶蛋白之一,也是在PKCα和突触后膜受体蛋白间起重要作用的衔接蛋白。PICK1分别由PDZ结构域、BAR结构域以及卷曲螺旋区和酸性氨基酸区组成。PICK1中的PDZ结构域和受体蛋白、转运蛋白、衔接蛋白的相互作用报道较多,BAR结构域则与支架蛋白、质膜等相互作用。PICK1在突触可塑性、神经递质传递、外周神经感觉、细胞生长和黏连等方面发挥重要作用。本文对PICK1的结构和功能进行综述。     

Overexpression of Protein Disulfide Isomerase in Aspergillus

One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzaeα-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale. Received: 26 April 2000 / Accepted: 30 May 2000     

The AMP-Activated Protein Kinase Is Involved in the Regulation of Ketone Body Production by Astrocytes

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.     

Mitochondrial Creatine Kinase: Properties and Function

This review describes properties of mitochondrial creatine kinase from heart and skeletal muscle studied in the author's group at the Department of Biochemistry of Moscow State University. The results are compared to the data in the literature. The author's point of view on the physiological role of mitochondrial creatine kinase is presented.     

Mitochondrial tRNA gene clusters in Aspergillus nidulans: Organization and nucleotide sequence

The organization of tRNA genes on the circular 32 kb mitochondrial genome of the ascomycete Aspergillus nidulans has been studied by gel transfer hybridization and by DNA sequencing. Most of the tRNA genes are tightly clustered within two regions (1 kb each) flanking the split gene for the large ribosomal subunit RNA. The upstream cluster contains nine genes, the downstream cluster eleven genes. The twenty tRNA genes are on the same strand as the two rRNA genes and are separated from each other by AT-rich spacer sequences, usually consisting of only a few nucleotides. Two tRNA genes (leul and ala) are joined end to end. The occurrence of two tRNA^Gty genes is the first exception to the observation that in mitochondria all four-codon families are read by a single tRNA. Both genes are adjacent and show extensive sequence homology, suggesting relatively recent origin by gene duplication. The product of glyl has a U in the wobble position as do all other tRNA gene products specific for four-codon families, whereas the gly2 product, which has a rare A in the same position, should read only the codon GGU. The products of metl and thr have an A and G in positions 18 and 55, respectively, like the mitochondrial tRNA^fMet and tRNA^Thr of Neurospora crassa. Other unusual features are the replacement of the invariant G-C pair at positions 53 and 61 by A-T in met2, glyl and gly2, the replacement of the invariant T at position 8 by A in phe and G in pro and the deletion of a nucleotide at position 9 in ser2.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.